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Utilizing the transporter associated with antigen processing (tap) to enhance immune responsiveness to… Alimonti, Judie Barbara 1999

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UTILIZING T H E TRANSPORTER ASSOCIATED WITH A N T I G E N PROCESSING (TAP) TO E N H A N C E I M M U N E RESPONSIVENESS TO CANCERS A N D VIRUSES by Judie Barbara Alimonti B.Sc, The University of British Columbia, 1991 A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies D E P A R T M E N T OF MICROBIOLOGY A N D I M M U N O L O G Y and T H E BIOTECHNOLOGY L A B O R A T O R Y We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH C O L U M B I A October 1998 © Judie Barbara Alimonti, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT For cytotoxic T lymphocytes (CTL) to recognize infected, mutated, or cancerous cells, an antigenic peptide must be presented on the cell surface by the major histocompatibility complex I (MHC I). The majority of peptides bound to MHC I arise from the cytosolic degradation of proteins. These peptides are translocated by transporters associated with antigen processing (TAP), into the endoplasmic reticulum (ER) where they bind with MHC class I before proceeding to the cell surface for recognition by CD8+ T lymphocytes. In TAP deficient cell lines the MHC class I surface expression is low resulting in poor recognition by T cells. Therefore TAP is important in the loading of MHC class I with peptide, and this thesis examines TAP's role in antigen processing and presentation and its effect on CTL mediated killing. Three aspects are considered: 1) the use of TAP as an adjuvant in vaccines, 2) the involvement of TAP in secreting antigen which sensitizes cells to CTL killing, and 3) restoring TAP in TAP deficient cancer cells to improve immune recognition and destruction of the tumour. CTL play an essential role in eliminating viral pathogens, and adjuvants can promote the induction of CD8+ CTL when injected along with antigenic peptide. The first study demonstrated a > 4.8 X increase in the frequency of specific CTL when TAP was included in a vaccine containing a cytotoxic peptide. Thus TAP can act as an adjuvant to enhance an immune response to a viral cytotoxic epitope. TAP is the major transporter of peptides for MHC class I assembly in the ER lumen. The second section in this study shows that there is an alternative fate for ER lumenal peptides other than binding to MHC class I. The peptides can be actively ii secreted from the cell through the constitutive secretory pathway in a TAP dependent manner. Furthermore, the secreted peptides can sensitize surrounding cells to be recognized by CTL. This demonstrates that CTL recognition of neighbouring uninfected cells presenting viral antigen involves TAP. Some cancer cells, like CMT.64, escape CTL mediated destruction due to low surface concentration of MHC class I. CMT.64 cells transfected with TAP restores viral antigen presentation to CTL in vitro. The final section of this thesis explores the role of TAP in restoring antigen presentation and immune recognition of cancer cells in vivo. Although TAP functions as a heterodimer (TAP1 and 2), re-expression of TAP1 in CMT.64 improved survival of tumour bearing animals dramatically. Furthermore, a Vaccinia Virus containing TAP1 could be used as a form of cancer therapy in CMT.64 burdened mice. These findings argue that T cell recognition of the tumours increases with the addition of TAP. Collectively, the addition of TAP to either wild type cells, and TAP deficient cells increased immunogenicity. This supports a role for TAP in the CTL mediated recognition of both viruses and cancers, and the use of TAP in the development of vaccines, or cancer therapeutics. iii Table Of Contents Abstract » Table of Contents iv List of Tables viii List of Figures ix Acknowledgments xi Dedication xii 1. General Introduction 1 1.1 The Specific Immune Response 3 1.2 The Major Histocompatibility Complex 6 1.2a MHC genetics 7 1.2b Structure and Biosynthesis of the MHC antigens 9 1.2c Antigen Presentation 12 1.3 Antigen Processing 14 1.4 TAP transports peptides into the ER 16 1.4a TAP structure and function 18 1.4b Peptide specificity of the TAP complex 20 1.5 Obj ectives and Approaches 21 2. Material And Methods 25 2.1 Molecular Biology Techniques 25 2.1a Plasmids and Bacterial Strains 25 iv 2.1b Oligonucleotides 26 2. lc Recombinant DNA Techniques 26 2.2 Cellular and Protein Techniques 27 2.2a Tissue Culture 27 2.2b Western Blots 28 2.2c Generation of Effector Cell Populations 30 2.2d Cytotoxicity Assay 31 2.2e Peptide Transfer Assay 31 2.2f Limiting Dilution Analysis 32 2.2g Peptides 33 2.2h Animals 33 2.3 Viral Techniques 33 2.3a Viruses 33 2.3b Recombinant Vaccinia Virus Construction 34 2.3c Selection of the Recombinant Vaccinia Virus 36 2.3d Southern blotting of rVV clones 37 2. 3e Purification of VV Stocks 37 2.3f Titration of Viruses 38 2.4 Statistics 39 3. Construction of Recombinant Vaccinia Viruses 40 3.1 Introduction 40 3.1a Vaccinia Viruses as Vectors 41 v 3.2 Results 42 3.2a Construction of VV-rTAPl, VV-NP, and VV-pJS5 42 3.2b Functional analysis of VV-NP, VV-TAP, and VV-pJS5 45 3.3 Discussion 49 4. Using TAP as an adjuvant 51 4.1 Introduction 51 4.1a Vaccine development 51 4.1b Subunit vaccines 53 4.1c Adjuvants 54 4. Id TAP as an adjuvant 56 4.2 Results 57 4.2a VV-NP as a model subunit vaccine 57 4.2b TAP enhances a specific immune response 59 4.3 Discussion 66 5. TAP is involved in surrogate antigen presentation 68 5.1 Introduction 68 5.2 Results 69 5.3 Discussion 76 6. rTAPl improves CTL recognition of CMT.64 cancer cells in vivo 78 6.1 Introduction 78 6.1a Cancer Phenotype 78 vi 6. lb Antitumour effector mechanisms 80 6. lc The CMT.64 cell line 82 6.2 Results 84 6.2a The TAP complex improves immune recognition in vivo 84 6.2b The nature of tumour recognition 94 6.2c Utilizing TAPs in cancer therapy 100 6.3 Discussion 108 7. General Conclusions 114 7.1 Viral antigen presentation 114 7.2 Tumour antigen presentation 118 8. Nomenclature 122 9. Bibliography 124 vi i List of Tables Table 1: Cell lines used in this thesis 29 Table 2: Viruses used in this thesis 35 Table 3: Summary of some common adjuvants 56 Table 4: Altered HLA class I phenotypes in tumour tissue 79 Table 5: Surface MHC class I expression by CMT.64 and CMT.neo 86 Table 6: Comparison of the tumor number and size in mice injected with CMT.64, CMT.neo, and CMT1.4 92 Table 7: Comparison of surface MHC class I expression with TD 5 0 in CMT:TAP transfectants 96 viii List of Figures Figure 1: Structure of the TcR and MHC 5 Figure 2: MHC Class I biosynthetic pathway 11 Figure 3: TAP complex structure 19 Figure 4: Construction of pJS5-rTAPI 43 Figure5: Construction of pJS5-VSVNP 44 Figure 6: rTAPl expression in a VV-rTAPl infection of CV-1 cells 47 Figure 7: Determination of VV-NP expression and VV-TAP function 48 Figure 8: Immune response to varying VV-NP dosage 58 Figure 9: Specificity of splenocytes from VV-NP immunized mice 60 Figure 10: Effect of TAP on VV-NP immunization 62 Figure 11: Effect of TAP on a low dose immunization 63 Figure 12: TAP does not augment VSV antigen presentation in RMA cells in vitro 65 Figure 13: VSV NP can be transferred to and sensitize uninfected RMA cells in a TAP-dependent manner 71 Figure 14: BFA inhibits peptide transfer 74 Figure 15: Emitine and azide inhibit peptide transfer 75 Figure 16: Comparison of the survival of mice injected with CMT.64 or the vector only control CMT.neo 85 Figure 17: rTAP12 transfected CMT.64 cells do not survive longer ix 3 than the TAP deficient CMT.64 cell 88 Figure 18: Viral presentation by CMT.64 transfected with single rTAPs 89 Figure 19: Improved survival of mice receiving rTAPl transfected CMT.64 91 Figure 20: Comparison of CMT:TAP transfectants with CMT.neo 93 Figure 21: Specificity of splenocytes from mice injected with CMT.neo or CMT1.4 97 Figure 22: rTAPl does not improve CMT.64 recognition in nude mice 99 Figure 23: Immunization with the CMT:TAP transfectants 101 Figure 24: VV-rTAPl therapy of CMT.neo burdened mice 104 Figure 25: Specificity of splenocytes from CMT.neo + VV-pJS5 mice 106 Figure 26: Specificity of splenocytes from CMT.neo + VV-rTAPl mice 107 Figure 27: Possible model for TAP adjuvant effect 116 x Acknowledgments Although this thesis represents my work I feel that the scientific process is a group endeavor. The researchers and technicians I have worked with have all contributed to my knowledge of science, aided in my experiments either directly or indirectly, and provided a friendly atmosphere to work in. Specifically, I would like to thank Dr. Wilfred Jefferies who provided enthusiasm for science, a great knowledge of immunology, and insightful suggestions on my project and science in general. UBC is a beautiful campus, but it is the people who helped to make the work enjoyable and interesting. The diverse scientific community of the Biotechnology Laboratory was instrumental in introducing me to varied concepts and techniques I otherwise would not have been exposed to. The Microbiology and Immunology Department has many devoted scientists who are also interested in the students they teach. In particular my committee members, Dr. D. G. Kilburn, Dr. W. R. McMaster, and Dr. M. Gold provided the critical and supportive comments to keep me on track. Doug Carlow was always very helpful and never turned me away when I needed specific expertise in immunology. I am grateful to both UBC and NSERC for their support which was instrumental to continuing my studies. Many individuals were helpful along the way and deserved to be mentioned. Dr. P. McAlpine gave me my first experience in a laboratory environment which first sparked my interest in research. Subsequently, I worked in the Cellulase lab for Dr. D. Kilburn, and Dr. R.A.J. Warren who provided a wonderful atmosphere to work in. A special thanks to Dr. Kilburn who always watched over me even after I left the lab. The people in the Cellulase lab who were responsible for teaching me the research techniques early in my career were Zahra Assouline, Emily Kwan, and Edgar Ong. There are many people in the Jefferies lab whom I am indebted to and would like to mention. Gaba, Malcolm, and Cachou were always supportive and helpful in the scientific process. A very special thanks goes out to Gregor Reid who was invaluable in helping me with my degree. When I was injured I was appreciative to have several people help me out including Gregor, Jacqueline, Brandie, Daphne, and Greg. Thank you Willem for your patience and expertise with the large number of mice which I was always having you look after. The remaining people in the lab have also been important in many ways deserved to be mentioned and include Alan, Jaana, Iku, Forrest, Mike, Ian, Roger, Alex, Joe, Cyp, Renee, Gerry, and Qian-Jin. Finally, I wish to acknowledge and thank my father Nick, my brother Keith, and sisters Nancy, and Cathy who have always been supportive and helped me out whenever or however they could. My mother, Sheila, was not able to see my achievement but her teachings helped me make it through this journey. Also, thank you to the Giesbrecht family who has always provided support and encouragement over the years. XI Dedication To my loving husband Alan Giesbrecht Thank you for your love and support. Your were always there to make me laugh, and pick me up when times were tough. I will never forget the sacrifices you made for me. xii 1. General Introduction Medical researchers have been studying disease for centuries in order to decipher the etiology of disease, discover why some people are unaffected and to determine the causes of varying diseases outcomes. There appear to be two causes of disease; one in which the pathology either arises from within due to genetic mutations, and one that is caused by external pathogens such as viruses or bacteria. Attempts to find cures and treatments have been a long and arduous journey, with successes realized for many infectious diseases (1). However, we have taken but one tiny step. We are still faced with tuberculosis, malaria, AIDs, cancer and other diseases that plague people in the late twentieth century (2,3). The search for cures has encompassed many disciplines, one of which has been immunology. With the advent of molecular biology the study of the immune system has gone beyond the realm of Aesop's fables and begun to provide some interesting information on how the body combats external pathogens, as well as how it recognizes and eliminates various types of cancers. Malignancies provide a challenge to modern immunologists since the tumour is derived from our own cells and the immune system is mainly tolerant to self components. The challenge is to provoke the immune system to destroy the cancer cell while leaving the rest of the body unaffected. A vast range of different tumours exist and what may work for one type may be ineffective for another. Research is beginning to discover some shared characteristics in the different types of tumours. Most notably, that many have viral or mutated self proteins which can be utilized to distinguish the malignant cell from the rest of the body. However, many lack the ability to present 1 antigen to cytotoxic T lymphocytes (CTL) so the altered proteins can never be detected (4,5). As we learn more about the various types of tumours we can begin to devise strategies to combat them. The search for a cure for diseases caused by external pathogens presents a different challenge from fighting cancers. Throughout history there are many documented cases of viral or bacterial epidemics that have been prevented by vaccination (6). However there remain many diseases in which this is not as yet possible. Some pathogens have evolved mechanisms to evade the hosts immune system such as antigenic variation of coat proteins, and acquisition of host antigens in their viral coat (7,8). While whole pathogen vaccination has worked, it can be unsafe due to the inactivation methods used, as incomplete killing of viruses in vaccines has resulted in causing the disease they were designed to prevent (1). To counteract this problem, the use of subunit vaccines has been explored where only the immunogenic portion of the pathogen is injected (9). This requires an in depth knowledge of the components of the organism to determine which part is most protective. Understanding a pathogen or disease process is very important, the knowledge of how the immune system reacts to and eliminates foreign agents provides us with insights into how to manipulate the immune response to treat tumours, and viral or bacterial diseases. The elimination of pathogens by the body is a learning process. Upon first encountering a pathogen, the non-specific early phases of the immune response predominate, in which macrophages can engulf and digest microorganisms. If the pathogen persists then the adaptive immune system is activated and recruits lymphocytes specific for the pathogen. In addition to fighting the pathogen, the 2 adaptive system establishes specific immunological memory such that following reinfection with the same agent, elimination of the pathogen is more rapid. Understanding how specific memory is achieved is the key to developing strategies that prevent diseases from being established. 1.1 The specific immune response One part of the immune system that recognizes and neutralizes foreign or 'non-self antigens is the adaptive immune response. This response includes B and T lymphocytes which possess antigen specific receptors for the recognition of extracellular or intracellular pathogens. The ability to detect a vast number of pathogens comes from the immense diversity of antigen receptors on B and T lymphocytes. Collectively, the immune system components can specifically recognize millions of different antigens, yet the lymphocytes that detect one particular antigen make up a very small proportion of the total lymphocyte population. The immune system is able to maintain diversity yet expand enough cells to mount an effective defense through a process called clonal selection. This involves activating and expanding the specific lymphocyte that encounters the antigen so that many specific cells are made from the original clone (10,11). The two arms of the adaptive immune system are the humoral and the cell-mediated immune responses. The humoral arm produces antibodies that bind to and neutralize extracellular pathogens and their toxins. B lymphocytes are the primary effector cells of this immune response and secrete specific antibodies that bind antigens and neutralize them or target them for destruction by other cells. The T helper 3 lymphocytes also participate in the humoral response through the production of cytokines that stimulate antibody production by B cells. There are two classes of T helper lymphocytes designated Thl and Th2. The Thl lymphocyte primarily mediates an inflammatory response, whereas the Th2 lymphocytes are responsible for the activation of the B lymphocytes. The cell mediated immune response controls intracellular pathogens or diseased cells through a cytotoxic mechanism mediated by T lymphocytes. Since this thesis is concerned primarily with the cytotoxic response to viruses and tumours, the remaining focus will be on the cell-mediated cytotoxic T cell (CTL) response. Essentially, when a CTL recognizes a foreign antigen or mutated self peptide on the surface of another cell, it sees the target cell as infected and proceeds to kill it. The interaction between the diseased cell presenting the antigen and the CTL involves the major histocompatibility complex (MHC) on the antigen presenting cell (APC) and the T cell receptor (TcR) on the CTL (figure 1A,B). The antigen generally undergoes processing inside the cell so that the TcR recognizes short fragments of the antigen in the peptide binding pocket of the MHC (12,13). Antigen recognition by T cells is MHC-restricted, meaning that a given T cell will recognize the antigen only when its peptide is bound to a particular MHC molecule (14). The TcR is comprised of two different polypeptide chains a and p, or on rarer cells, y and 8, bound together by a disulphide bond (figure 1A). The variable region of the TcR is the product of gene rearrangement, whereby many smaller gene segments are spliced together to form the coding region for a single chain of the receptor (12,15). It is this gene rearrangement that provides the T cell population with a diverse 4 B 2C TCR 2C TCR c Figure 1: Structure of the TcR and MHC class I A) Structure of a mouse ap TcR heterodimer. B) Variable domain of a mouse ap TcR heterodimer with the H-2K" MHC class I containing a peptide. C) Structure of MHC class I, and its peptide binding pocket with a peptide. 5 set of TcRs while retaining a high degree of specificity. Unlike antibodies which can bind soluble molecules, the TcR only recognizes the membrane bound MHC complexes on the APC. It is the hypervariable regions of the TcR which form the contact points with the MHC:peptide complex. The T cell has two co-receptor molecules, CD4 and CD8, which bind to invariant portions of the MHC class II and I respectively. During antigen recognition, the co-receptors act by making TcR signaling more efficient and providing a 100-fold increase in the sensitivity of T cells to antigen (16). I. 2 The Major Histocompatibility Complex The MHC complex is expressed on the surface of cells and was originally identified by its involvement in transplantation rejection (17,18). The MHC has evolved to allow the host to discriminate between self and non-self, thereby eliminating foreign, and non-self elements that the host detects (19). Essentially the MHC antigens are responsible for the display of peptides to the antigen receptors found on T lymphocytes. The two main types of MHC antigen on the cell surface, class I and class II, are distinct in structure, synthesis, distribution, and function. Although there are exceptions, we can say in general, that the MHC class II is responsible for presenting extracellular antigens, whereas MHC class I primarily present peptides that are synthesized within that cell. The two classes of MHC are recognized by distinct T lymphocyte subsets which results in the stimulation of disparate effector mechanisms (16). Class II antigens are found mainly on cells of the immune system, such as the professional antigen presenting cells which include dendritic cells, activated B cells, 6 and macrophages (m§) (20). The CD4+ T lymphocytes recognize MHC class II and can be either the Thl or Th2 phenotype. Stimulation of the Th lymphocyte influences the immune response through the release of cytokines which activate m<j), enhance CTL development, influence B cell activation and Ig isotype switching, along with many other functions. The Th cells are instrumental in the development and amplification of an immune response. MHC class I antigen is generally recognized by CD8+ T lymphocytes which are primarily cytotoxic (16). MHC class I antigens are expressed on most nucleated cells, with some exceptions, such as neuronal cells (21,22,23). Due to the almost ubiquitous expression of MHC class I, the CD8+ CTL are able to survey the entire body and kill any diseased or infected cells to prevent further pathogenesis; a process referred to as immunosurveillance. These would include cells displaying foreign peptides when infected by intracellular pathogens like viruses. Or, it could be a tumour cell which is synthesizing a 'foreign' altered self protein. 1.2a MHC genetics The polygenic MHC locus is generally subdivided into three regions named class I, II, and III (24). These loci encode for antigen presenting molecules (MHC class I and II), proteins involved in antigen processing (i.e.: LMP and TAP), as well as other proteins having specialized functions in immunity (i.e.: complement proteins, and tumour necrosis factor). The location of the MHC locus in mice is the H-2 region on chromosome 17, whereas the human counterpart is called the HLA region and is encoded on chromosome 6. The class I and II regions encode the classical major 7 histocompatibility antigens, MHC class I and MHC class II. The class II genetic loci in the mouse are called I-A and I-E, and in humans are DR, DP, and DQ. There are three classical loci for class I antigens, named K, D, L in the mouse, and A, B, and C in humans (25). Although there are three sets of genes encoding MHC class I molecules, the actual number of different MHC expressed on the cell is higher due codominant gene expression and genetic polymorphism which has given rise to a large number of alleles for each gene. For example in humans, each of the MHC class I A and B genes have approximately 100 alleles although the HLA C gene appears less polymorphic. Most individuals are heterozygous at each genetic locus so there could be a total of six class I gene products in an individual that can be involved in presenting antigens to CTL. For MHC class II in humans, HLA-DP and DQ each have an a and P chain gene. However, the HLA-DR cluster contains an extra p chain gene whose product can pair with the DR a chain. Therefore, three sets of genes can give rise to four types of MHC class II molecules, so eight different molecules can be expressed on a cell at one time. The MHC in humans is one of the most polymorphic cluster of genes, and affects antigen recognition by T cells, rejection of tissue grafts and susceptibility to immunological disorders. The importance of the MHC in distinguishing self was obtained from work on tissue transplantation in mice (17,18). It was discovered that the MHC genes controlled tissue compatibility between members of the same species. A tissue transplant to another individual differing in expression of the MHC genes resulted in rejection of the graft. Tissue transplants between inbred mice with identical MHC alleles at each locus were not rejected. The polymorphism in MHC also 8 Influences the ability of the immune system to recognize and mount a response against foreign pathogens. As will be discussed later the T cells can only recognize an antigen within the context of a particular MHC allele. Overall, the polymorphism of the MHC molecules allow the immune system to distinguish self from the vast number of pathogens that are encountered. 1.2b Structure and biosynthesis of the MHC antigens The structure of MHC molecules has been elucidated. Both MHC class I and II molecules are transmembrane glycoproteins found on the plasma membrane. The different peptides displayed by the two MHC antigens are due to their disparate biosynthetic pathways and structure. To begin with, the MHC class II complex is comprised of two non-covalently associated proteins, a and p. Each subunit contains two disulphide linked extracellular domains, a transmembrane domain and a cytoplasmic tail. The highly polymorphic peptide binding region is formed by the al and pi domains, and binds peptides 10-34 residues in length. During assembly, the a and p chains bind to the invariant chain (Ii) to form a nonameric complex containing three copies of each protein. This complex is unable to bind ER resident peptides due to Ii covering the peptide binding pocket (26). The intracellular targeting sequences in the Ii chain directs the complex to the cell surface via the endocytic pathway. When the complex reaches the MHC class II containing compartment (MIIC) the Ii is cleaved leaving a class II Ii peptide (CLIP) fragment in the binding cleft. CLIP is removed by the DM protein, which is a class II related molecule found in the MIIC. Removal of 9 CLIP from the peptide binding pocket allows the empty MHC class II complex to bind the peptides in the endocytic compartment prior to transport to the cell surface (27,28). Since MHC class II cannot bind to peptides in the ER lumen it tends to acquire peptides from proteins that are endocytosed. This means that the majority of peptides bound to MHC class II are derived from extracellular sources. In contrast to the MHC class II, the MHC class I complex consists of a heavy chain (HC), a peptide, and a non-MHC encoded beta 2 microglobulin (P2m). The HC has a cytoplasmic tail, a transmembrane domain, and three (al, a2, a3) extracellular domains (figure 1C). Both the a2 and a3 domains are stabilized by intradomain disulfide bonds. The polymorphic peptide binding pocket is created from residues in both the al and a2 extracellular domains and binds peptides 8-10 residues in length. The MHC class I HC is not associated with a peptide when it is translocated into the ER and cannot proceed to the cell surface until it changes its conformation by binding both peptide and p2m (figure 2). The assembly of the MHC class I-peptide complex in the ER is a multistep process involving several proteins (29,30, [reviewed in 31]). Calnexin associates with HC soon after it is synthesized and retains incompletely assembled HC in the ER. In the mouse, p2m can bind the HC while the HC binds calnexin. However, in humans p2m displaces calnexin and then the HC-p2m complex binds to calreticulin. Calreticulin associated class I molecules can then associate with the transporter associated with antigen processing (TAP) via a protein called tapasin (32). TAP is responsible for transporting peptides from the cytoplasm into the ER where they can bind to MHC class Lp2m complexes. Upon successful binding of a 10 Plasma Membrane Constitutive secretory vesicle Golgi Complex ER M H C I p 2m calreticulin heavy chain cytoplasm t cytoplasmic protein proteasome peptides Figure 2: MHC class I Biosynthetic Pathway MHC I heavy chain is cotranslationally inserted into the ER membrane and is bound by calnexin until MHC I complexes with P2m a n Q ( calreticulin. A multimeric complex is formed between class I-p2m-calreticulin and TAP via a tapasin bridge. TAP transports peptides, derived from proteolysis of cytoplasmic proteins, into the ER. The peptides combine with MHC class I heavy chain~p2m, before the MHC classl-peptide complex is transported to the cell surface through the constitutive secretory pathway. 11 peptide, the MHC class I: p^ n^ peptide complex is released, allowing transport to the cell surface for recognition by T lymphocytes. Unlike MHC class II, MHC class I does not go to through the endocytic pathway but instead passes through the ER and Golgi and then to the cell surface via the secretory pathway (33). It is clear from the differing structures and biosynthetic pathways of the two MHC that they may interact with separate peptide pools. The peptides that MHC class I binds in the ER originate in the cytosol and in general are derived from endogenously synthesized proteins. The remaining sections will focus on MHC class I which binds endogenously-produced peptides, as these are more relevant for the immune response against tumours and viral infections. 1.2c Antigen presentation The function of the MHC is to deliver peptide fragments of an antigen to the cell surface where the peptide:MHC complex can be recognized by T cells. Presentation of non-self peptide will initiate a response via the TcR and its coreceptors. So, it is the structure of the displayed MHC-peptide complex which influences the effector mechanisms of the immune system. There are several factors which affect the particular peptide displayed, aside from the distinct peptide pools that each MHC encounters from alternative maturation pathways. Further peptide selection occurs due to differing peptide pockets (figure 1C). The ends of the MHC class I binding pocket form a closed structure, therefore the size of the peptides are limited to 8-10 amino acids in length. In contrast, MHC class II 12 binds various lengths of peptide due to an open ended peptide pocket (34). The peptide lies in an extended conformation with contacts between main chain atoms and amino acid side chains that line the groove. The side chains of the polymorphic residues lining the peptide binding pocket determine the peptide binding properties of the different alleles of MHC molecules (35). This polymorphism is critical to antigen recognition as T cells recognize peptides bound by a particular MHC allelic variant, and will not recognize the same peptide bound to other MHC molecules (14). In the case of MHC class I, the pocket has defined sections, termed A to F, in the cleft which interact with various amino acids of the peptide (36,37). In sections A and F at either end of the peptide groove are highly conserved residues that serve as anchoring sites for the amino- and carboxy-terminal ends of the peptide (38). The other residues in the cleft are variable so MHC alleles will differ in the location and shape of the pockets in the cleft which can accommodate certain amino acid side chains of the peptide. It is the variable pockets that are believed to determine the specificity of the peptide binding (37). Pocket depth or capacity to bury the side chains of bound peptide has a direct impact on peptide binding. For example, in H-2K" the deep pocket C determines sequence specificity of the peptide. Section C accommodates a tyrosine or phenylalanine residue whereas the shallow pockets D and E are less critical for peptide selection. Residues that fit optimally into these pockets occur with high frequency in specific positions in the peptide associated with MHC class I. In general, the peptide has two to three anchor residues for binding to MHC. The anchor residues differ for peptides binding different MHC molecules but are 13 similar for all peptides binding to the same MHC molecules (39). The particular set of anchor residues that allow binding to a given MHC class I molecule is called a sequence motif (40). The octameric Vesicular Stomatitis Virus (VSV) N peptide (R'G2Y3V4Y5Q6G7L8) contains the H-2Kb restricted motif (41). This specific motif requires a tyrosine or phenylalanine at peptide residue 5 or 6, as well as a leucine at residue 8 or 9. The tyrosine at residue 5 in VSV N peptide fits into the deep pocket C of the H-2Kb peptide groove, with the 8th leucine residue fitting into pocket F. In 1990 there were only three MHC ligands known, however the list has now grown to several hundred. Knowledge of the ligands will help to determine the specific sequence motifs required for binding to MHC and therefore allow better identification of potential epitopes within a protein. 1.3 Antigen Processing The majority of peptides presented by MHC class I are derived from proteins synthesized and then degraded in the cytoplasm. The complex believed to be responsible for creating peptides in the cytosol is the multicatalytic protease complex called the proteasome (42). It is a 28 subunit cylindrical structure consisting of four stacks of 7-membered rings. The two outer rings each contain seven different a-type subunits, and the two inner rings each contain seven different P-type subunits. The proteasome is involved in both the ATP-dependent ubiquitin-dependent, and ubiquitin-independent pathways of protein degradation. One line of evidence suggesting the involvement of the proteasome in antigen processing came from experiments demonstrating that ubiquitination could increase the efficiency of peptide generation 14 (43). The proteasome exists in several distinct forms which contain different subunit components. The two primary complexes are the 20S and 26S proteasomes which possess trypsin, chemotrypsin, peptidylglutamyl-peptide bond hydrolase, and additional serine protease activities (44). The exact proteolytic subunits involved in the protease activities remain unclear. However, to date it is known that the a-type subunits are non-catalytic, whereas three of the seven (3-type subunits (delta, LMPX and Z) have catalytic activity (45,46,47). It has been determined that the 20S proteasome is a precursor of the 26S proteasome. The 20S proteasome combines with two regulatory complexes, each composed of fifteen different proteins, to form the 26S proteasome. The extra subunits of the 26S proteasome confer a different substrate specificity allowing it to degrade ubiquitinated proteins and other proteins in a ATP-dependent manner (16,48-50). Further support for proteasome involvement in antigen processing came from the discovery that two of three IFN-y inducible genes encoding LMP2 and LMP7, were in the MHC class II genomic region and are likely coordinately regulated with the TAP genes (51). Treatment with IFN-y results in a loss of constitutive proteasome components delta, LMPX and Z, which are replaced by the upregulated components LMP2, LMP7 and MECL (47,52). This stimulates the trypsin and chemotrypsin activities and depresses the peptidylglutamyl-peptide hydrolyzing activity (46,52,53). While early studies had shown that LMP2 and LMP7 were not essential for antigen presentation by class I, more recent results suggest they enhance the specificity of the proteasome complex by increasing cleavage after hydrophobic and basic residues (46,54,55). This correlates well with the characteristics of the peptides that bind to class I molecules (56). 15 1.4 TAP transports peptides into the ER The transport of peptides into the ER is performed by the TAP complex. Evidence for the role of the heterodimeric TAP complex in peptide transport into the ER was obtained from studying several cell lines deficient in MHC class I processing. These included the TAP1 and TAP2 deficient human cell lines T2, and .174 (64,65), as well as the TAP1 deficient .134 (66), and the TAP2 deficient murine cell line RMA-S (67,68). In these mutant cell lines, the MHC class I proteins are retained in the ER resulting in low cell surface levels and the inability to present intracellular antigens efficiently. In the case of T2, the cell synthesizes normal levels of MHC class I heavy chains but they fail to assemble with p2m and peptide, resulting in the slow transit of empty, labile class I molecules to the cell surface (69,70). However, their stability on the surface can be enhanced by the addition of exogenous antigenic peptides (71). The low supply of peptides into the ER was shown to be the cause of the defect as transfection of the TAP molecules into the mutant cell lines increased surface MHC class I expression, as well as allogeneic and viral antigen presentation (66,72-74). The TAP1' knockout mice have reduced numbers of MHC class I and consequently have very few mature CD8+ T cells (75-80). These results demonstrate that the TAP complex is essential, through its role in supplying peptides, for expression of high levels of stable class I molecules on the surface of cells. The evidence demonstrating transport of peptide into the ER by TAP has come from in vitro assays. It was shown in microsomes from wild type mice that peptide accumulated at a greater rate than in microsomes from TAPI"'" mice (76). Furthermore, 16 the accumulation was found to be specific and dependent upon the availability of ATP. Another in vitro assay measured peptide translocation by semi-permeabilizing the plasma membrane of cell lines and adding ATP along with iodinated peptides containing a N-glycosylation site. Only the peptides that were transported into the ER would be glycosylated so the level of peptide transport in normal and TAP deficient cells could be measured by adsorption of the glycosylated peptides to concanavalin A-sepharose beads (81). Both of these in vitro assays confirm that TAP transports the peptides into the ER in an ATP dependent manner. Hence TAP could act in a selective role, from a pool of peptides. Other sources of peptides exist including the proteins degraded in the ER, and the trimming of signal sequences (57-59). In T2 cells which lack the TAP complex, the isolation of peptides from MHC class I demonstrated that the peptides were derived from signal sequences and contained only seven dominant peptides as opposed to several hundred for TAP expressing cells (60). There are also examples of a TAP independent peptide transport, as was seen with the ability of T2 cells to present Sendai virus peptides to CTL (61,62). The presentation was shown to be resistant to brefeldin A, an inhibitor of protein secretion, suggesting that the viral peptide did not bind to MHC class I in the ER. The underlying mechanism has yet to be determined. However, because TAP knockout mice exhibit a profound lack of cell surface class I molecules, these TAP independent peptide sources must be minor contributors in comparison to the supply of peptides via TAP mediated transport from the cytoplasm (63). 17 1.4a TAP structure and function The TAP complex is a member of the ATP binding cassette (ABC) transporter family. Proteins in this family are found in many cells and are always involved in ATP-dependent transport of ions, sugars, amino acids, or peptides. Members of this family form complexes generally consisting of four domains that include two transmembrane and two ATP binding domains (82). In co-immunoprecipitation studies, the TAP complex has been shown to form a heterodimer consisting of TAP1 and TAP2 (83-85). As seen in figure 3, each TAP subunit contains multiple membrane spanning regions, and a cytoplasmic ATP binding region (86,76). The TAP complex has been localized primarily to the ER as well as to the cis-Golgi (84,87). It was suggested by several chemical crosslinking experiments that both subunits of the TAP complex are required for peptide transport to occur (86,88). In contrast, the TAP2 deficient cell line RMA-S can transport TAP dependent peptides, suggesting the possibility of a TAP1 homodimer (89,90). The presence of ATP is required for the transport of peptide into the ER but not for the binding of peptide to the TAP complex (80,91). The peptide binding region is found in the transmembrane domain closest to the ATP binding site. Other studies examining the specificity of peptide transport demonstrated two polymorphic regions in the hydrophilic loops protruding into the cytoplasm as being important for peptide specificity (92). It is interesting that the optimum size of peptide that the TAP complex transports is 8 - 13 amino acids which is similar to the size of peptide which the MHC class I typically binds (91,93). Transport of longer peptides can occur but with lower efficiency, while transport of peptides smaller than 8 amino acids has not been shown. 18 TAP1 TAP2 ER NH, NH, ATP ATP -COOH COOH Figure 3: TAP complex structure The TAP complex is a heterodimer composed of two proteins named TAP1 and TAP2. Each glycoprotein contains a transmembrane domain and a cytoplasmic domain. The transmembrane domain spans the ER membrane an estimated 6 to 8 times, and contains the residues responsible for peptide binding and transport. Both subunits are required for the formation of the peptide binding domain.The cytoplasmic domain contains the cytosolic ATP binding domain. Hydrolysis of ATP is needed for the translocation of peptides into the lumen of the ER. 19 TAP could contribute to peptide loading simply by raising the concentration of free peptide in the ER. However, TAP co-precipitates with MHC class I suggesting that TAP may play an active role in the assembly of class I (94,95). Newly assembled, but peptide deficient MHC class I HC-.p2m associates rapidly with TAP, either directly or through Tapasin (30,32). Release from TAP and transport to the cell surface only occurs when HC:P2m binds peptide. The association between TAP and MHC class I may increase the efficiency of peptide capture. The rate of release from TAP was found to mirror the transport of peptide loaded MHC class I molecules from the ER to the Golgi (94,95). This data suggests that the rate of MHC transport to the cells surface is dependent upon the efficiency of peptide transfer, by TAP to 'empty' MHC class I. 1.4b Peptide specificity of the TAP complex The structural characteristics of transported peptides have been partially elucidated. Aside from preferentially transporting peptides 8-13 amino acids long, the TAP transporters appear to have only a limited requirement for specific residues in peptides (96). Peptide transport assays, measuring the transport of peptides with various amino acid substitutions, demonstrated that the peptide's carboxy-terminal residues govern the substrate specificity of TAP (97). Mouse TAPs are the most stringent as they primarily transport peptides with hydrophobic C terminal residues. At the other end of the spectrum are the more promiscuous human TAPs, which are not affected by C terminal residues (98). While allelic forms of human and mouse TAP have not yet been shown to have significant effect on peptide selectivity, the rat alleles have demonstrated different preferences for peptides (99). 20 The rat TAP" heterodimer resembles the mouse TAP in specificity, whereas rat TAPa is more like the human complex (100,101). The two different forms of the rat transporter are based on two allelic versions of the rat TAP2 molecule, which differ by 25 amino acids, spread throughout the membrane spanning region (102). The difference in the rat heterodimers results in distinct pools of peptides available for binding to rat class I (RTlAa). The RTlAa molecule prefers peptides that have basic residues, therefore it only becomes efficiently loaded in the presence of the TAP3 complex (103). TAPs are able to transport a diverse set of peptides which would seem appropriate for providing a large repertoire of internal peptides for MHC class I to bind. If MHC class I is to provide a sampling of internal peptides to the CTL then it should have access to as many peptides as possible. However, it is clear that TAP does provide some length and amino acid restrictions on the peptides it transports into the ER. Therefore TAP could be providing some limitations on the peptide repertoire for MHC class I. 1.5 Objectives and approaches The immune system can recognize diseased or infected cells that display foreign or mutated self peptides presented by MHC class I. The TAP complex is responsible for supplying and loading MHC class I with peptides, so what is transported by TAPs will have an impact on the immune response (88). As a result, the TAP complex could influence the immune response in two ways. First, it could enforce a limited selection on the peptides which bind to and are displayed by MHC class I. Second, since the 21 TAP complex is the major conveyor of peptides to the ER, it is an essential component of the class I biosynthetic pathway (104). In the absence of peptides MHC class I cannot traffic efficiently to the cell surface. If the MHC class I cannot reach the cell surface, then they are unable to bind the TcR and activate T lymphocytes, thereby diminishing the immune system's ability to eliminate the diseased cell. Since the TAP complex is a vital component of the antigen processing and presentation pathway, the aim of this work was to examine if TAP could be used to increase the CTL-mediated immune response to specific antigens and thus have a possible role as a therapeutic agent. Three specific experimental models were examined in which the role of TAP was studied: 1) as an adjuvant in a vaccine model, 2) to determine the involvement of TAP in the secretion of viral peptides which can sensitize neighbouring cells to CTL lysis, and 3) to restore immune recognition and destruction of a TAP deficient cancer cell. In the attempt to find effective vaccines many types of carrier molecules and immune enhancing adjuvants are being investigated. In order to elicit a cytotoxic memory response to viruses, the primary goal is to get the cytotoxic peptide into the endogenous antigen processing pathway where it can be presented by MHC class I. The carriers and adjuvants that have been devised to date include the use of liposomes (105,106) or adding lipophilic tails (107) to peptides in order to deliver the antigenic protein into the cell's cytoplasm. Another method is the use of viral vectors which can infect a cell and then undergo transcription and translation in the cytoplasm thereby introducing the antigen into the protein processing pathway (108). In this study Vaccinia Virus (VV) was used as a viral vector to deliver the VSV CTL epitope along 22 with TAP into the cytoplasm of the cell. It was hypothesized that the limiting factor in MHC class I biosynthesis is the supply of peptides by the TAP complex. It was investigated whether the addition of TAP to a wild type cell can increase antigen presentation to CTL in order to determine if this enhances antigen specific cytotoxic responses. A model subunit vaccine was created, that contains the cytotoxic epitope for the vesicular stomatitis virus (VSV) N protein in Vaccinia virus (VV). The human TAP molecules in a VV vector was also used to test the effect of expressing TAP in a subunit vaccine. It was determined whether the addition of TAP increases the level of the immune recognition of the cytotoxic epitope. In a second set of experiments the effect of TAP on priming non- infected neighbouring cells with viral antigenic peptide was explored. TAP is the major supplier of peptides in the ER for MHC class I. ER lumenal peptides that cannot bind to or are in excess of MHC class I have been found to be degraded, bound to heat shock proteins (HSP), or transported back into the cytoplasm (93,109-112). However, the ER is part of the cell's constitutive secretory pathway so it is possible that peptides can also be secreted from the cell. It was determined whether peptide secretion occurs and also if TAP plays a role in this process. In order to examine this possibility a peptide transfer assay was used to see if viral peptides from infected cells could prime neighbouring uninfected cells to CTL lysis. The third model in which TAP was used to enhance an immune response is by expressing it in a tumour cell to determine if it can restore antigen presentation and therefore enhance the destruction of the cancer cell in vivo. The immune system is 23 involved in the defense against spontaneously derived aberrant cells. However, malignancies can develop in the presence of the immune system. One of the mechanisms that may allow the evasion of the immune response is the downregulation of MHC class I which would prevent the presentation of tumor antigens to T lymphocytes (113-116). In this study, the effect of TAP on increasing antigen presentation in cancers was investigated. This was demonstrated in two ways. First a TAP-transfected cancer cell was used in an in vivo mouse model to see if TAP would enhance the recognition and destruction of the cancerous cell. Finally, the use of a TAP as a form of cancer therapy in malignancies was explored, using an in vivo mouse cancer model. 24 2. Material and Methods 2.1 Molecular Biology Techniques 2.1a Plasmids and Bacterial Strains The plasmid pJS5 (obtained from Dr. B. Moss, NIH, Bethesda, USA) was a shuttle vector used first in bacteria to clone in the gene for rTAPl, or the minigene for amino acids 52-59 of the VSV N protein (VSV NP). The pJS5 plasmid containing the cloned gene was then transfected into mammalian cells for homologous recombination with wild type Vaccinia Virus (VV). The pJS5 plasmid contains an E. coli ampicillin resistance gene for plasmid selection in bacteria, as well as an E. coli guanine phosphoribosyl transferase (gpt) resistance gene for selection of recombinant VV (rVV) in cells. pJS5 contains two synthetic VV promoters in front of a multiple cloning site (MCS) where either rTAPl or VSV NP were cloned into, giving the respective plasmids pJS5-rTAPl and pJS5-VSV NP. The entire section including the two promoters, MCS, and gpt resistance gene is flanked by the 5' end of a thymidine kinase (tk) gene downstream, and the 3' end of the tk gene upstream, for homologous recombination into the tk gene of VV. The source of the rTAPl or 2 was the mammalian expression vector pHp Apr-1 neo containing the full length cDNA of rat TAP1, or TAP2 (generously given by Dr. G. Butcher, AFRC, Cambridge,UK.). The plasmids were amplified in the E. coli strain DH5aF' grown in Luria-Bertani (LB) medium or on LB agarose plates containing 50 p-g/ml ampicillin. 25 2.1b Oligonucleotides The two complementary oligonucleotides used to make the minigene for VSV N52-59 were synthesized using an Applied Biosystems automated DNA synthesizer model 380A at the NAPS unit (U.B.C., Vancouver, Canada). The oligonucleotides and the amino acids they coded for were as follows: SacI overhang Nhel overhang 1) 3'-TCGAG-TAC-TCT-CCT- ATA-CAG-ATG-GTT-CCG-GAG-ACT-CGATC-P 2) 5'- P-C-ATG-AGA-GGA-TAT-GTC-TAC-CAA-GGC-CTC-TGA-G Peptide start - arg - gly - tyr - val - tyr - gly - gly - leu - stop P = P03 After purification on Pharmacia nick spin columns, equimolar amounts of each oligonucleotide were annealed in ligase buffer (Boehringer Mannheim) for 2 minutes at 95°C before cooling slowly to room temperature. The oligonucleotides were designed to provide overhangs corresponding to cleaved restriction sites for direct ligation into the Nhel and SacI cut pJS5 vector. 2.1c Recombinant DNA Techniques All protocols for recombinant DNA work are described in Maniatis (117). Double-stranded plasmid DNA was prepared using Qiagen maxi-prep kit. The modifying and restriction enzymes for treating plasmids were used according to manufacturers recommended protocols. After restriction digest of the plasmid, the 26 DNA fragments were separated by TAE (tris-acetate EDTA) agarose gel electrophoresis and purified using the Glass Max kit (Gibco). The vectors and inserts were ligated with T4 DNA ligase (Boehringer Mannheim) followed by heat inactivation and n-butanol precipitation before transforming electrocompetent E. coli cells by electroporation according to manufacturers instructions (Biorad electroporator). Transformed cells were selected on LB-ampicillin plates (50 p.g/ml). Double stranded DNA sequencing was performed by the dideoxyribonucleotide chain-terminating method (118) using Amersham Sequenase Kit II. 2.2 Cellular and Protein Techniques 2.2a Tissue Culture The cell lines used in this thesis and their source are listed in Table 1. Cell lines not obtained from ATCC were generous gifts from the people mentioned. A brief description of the more important cell lines follows. The small cell lung carcinoma cell line, CMT.64, used in the cancer experiments originated spontaneously from the C57BL/6 mouse strain (119). All of the stable CMT.64 transfectants containing rTAP-1 (CMT1.4, CMT1.10), rTAP-2 (CMT2.1, CMT2.10), rTAPl and 2 (CMT12.12, CMT12.21), and the vector only control (CMT.neo) were created by Gregor Reid and Reinhard Gabathuler in the Jefferies' lab by transfecting CMT.64 cells with the rTAP cDNA in mammalian expression vector pH(3 Apr-lneo (89,120). All cell lines were grown in either DMEM or RPMI containing 10% heat inactivated (HI) fetal bovine serum (FBS), and penicillin/streptomycin (P/S). The exceptions were T2Kb, which was 27 grown in Iscoves medium containing 10% FBS, 2mM L-glutamine, and 0.5 p.g/ml G418 (Gibco, BRL). The Hela S3 cell line was grown in DMEM except for large scale spinner cultures which were grown using Joklik medium containing 7% FBS or horse serum (HS) and P/S. The cell lines used for culturing and titering VV were CV-1, Hela S3, and BS-C-1. Vero cells were used for culturing VSV. When adherent cell lines were confluent, the cells were dislodged with 0.25% trypsin (W/V) in phosphate buffered saline (PBS) containing 1 mM ethylenediaminetetraacetic acid (EDTA) for 3 minutes at 37°C, and then replated with fresh medium after dilution. Non-adherent cells were cultured at cell densities of 1 x 105 and 1 x 106 cells/ml. The cells were passaged by centrifuging the culture for 3 minutes at 314 x g (Beckman GP tabletop centrifuge) and resuspending the pellet in fresh medium. All cells were incubated at 37 °C in a humidified, 5 % C0 2 / 95 % air environment. The exception was the spinner flask cultures which were cultured in a non-C02 37 °C incubator. 2.2b Western blots For Western blots, 106 infected or non-infected CV-1 cells were lysed in 1 ml. PBS, 1% NP-40, ImM PMSF and incubated for 30 min. on ice, followed by centrifugation at 11,000 x g for 10 min at 4 °C. Using the BioRad Mini Protean system a portion of the lysate was separated on a 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and run at 200V for 45 min. (123). The samples were then blotted onto Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore) using a Tris-glycine-methanol transfer buffer system for one hour at 100V 28 Table 1: Cell lines used in this thesis Cell Line Description Source BS-C-1 African green monkey kidney cells ATCC CCL 26 CV-1 African green monkey kidney cells ATCC CCL 70 CMT.64 Murine small cell lung carcinoma L. M. Franks, Imperial Cancer Research Fund, London, UK CMT.neo CMT.64 transfected with vector only G. Reid, R. Gabathuler, W. A. Jefferies, UBC, Vancouver, Canada CMT1.4 & CMT1.10 two clones of CMT.64 transfected with rat TAP1 G. Reid, R. Gabathuler, W. A. Jefferies, UBC, Vancouver, Canada CMT2.1 & CMT2.10 two clones of CMT.64 transfected with rat TAP2 G. Reid, R. Gabathuler, W. A. Jefferies, UBC, Vancouver, Canada CMT12.12 & CMT12.21 two clones of CMT.64 transfected with rat TAP1 and 2 G. Reid, R. Gabathuler, W. A. Jefferies, UBC, Vancouver, Canada Hela S3 human epitheliod cervical carcinoma ATCC CCL 2.2 RMA Rauscher virus-induced murine leukemic cell line (ref: 121,122) P. Cresswell, Yale Univ., Connecticut, and H-G. Ljunggren, Karolinska Institute, Stockholm RMA-S RMA cell line was chemically mutagenized and selected for reduced MHC class I (ref: 121,122) P. Cresswell, Yale Univ., Connecticut, and H-G. Ljunggren, Karolinska Institute, Stockholm T2Kb human B lymphoblastoid cell line deficient in TAP and MHC class II, transfected with Kb P. Cresswell, Yale Univ., Connecticut, and H-G. Ljunggren, Karolinska Institute, Stockholm Vero African green monkey kidney cells ATCC CCL 81 29 (124). The membrane was then blocked in blocking buffer (3% BSA, 0.1% Tween-20 in PBS) overnight at 4°C, and washed once with wash buffer (PBS, 0.1% Tween-20) before adding the rabbit anti-rat TAP1 polyclonal antibody (D90) at a dilution of 1:1000 in PBS, 1% BSA, 0.1% Tween-20. The signal was amplified by incubating with biotin donkey anti-rabbit IgG (1:5000) for 1 hour and the blot was washed 3 times before adding streptavidin-horse radish peroxidase (SA-HRPO, Jackson, 016-030-084) at 1:5000 for 30 min. The membranes were developed according to the manufacturer's instructions for the enhanced chemiluminescence (ECL) detection kit (Amersham) and the blot exposed to X-ray film (Kodak XAR). 2.2c Generation of Effector Cell Populations Virus-specific CTL populations were generated by infecting mice intraperitoneally (ip) with 107 tissue culture infection dose (TCID) units of VSV or at the suggested plaque forming units (pfu) for VV-VSVNP and VV-TAP. CTL were collected on day 5 post immunization from the cervical lymph nodes (LN), or spleen and cultured in RPMI-1640 medium containing 10% HI HyClone FBS (Gibco), 20 mM Hepes, 2 mM L-glutamine, 0.1 mM essential amino acids, 1 mM sodium pyruvate, 50 uM p-mercaptoethanol (p-ME), and penicillin/streptomycin (henceforth referred to as RPMI complete medium). The LN cell suspensions were cultured at 4 x 106 cells/ml for 3 to 5 days in the absence of stimulation before being used in a CTL assay, whereas the splenocyte suspension was cultured for 7 days with peptide stimulation. Bulk populations of VSV-specific CTL were maintained by weekly restimulation with 1 uM 30 VSV N peptide (amino acids 52-59) plus pulsed irradiated (2200 rads) stimulator splenocytes. Irradiated stimulator cells and CTL were incubated together at a ratio of 4:1 in RPMI complete medium containing 20 units/ml hIL-2. Seven days later, this bulk population was used in a CTL assay. 2.2d Cytotoxicity Assays Target cells for the CTL assays were loaded with 5 lCr by incubating 106 cells with 100 uCi of 5 lCr (as sodium chromate, Amersham) in 250 ul of CTL medium (RPMI-1640 containing 10% HI FBS, 20 mM Hepes) for 1 hour. Following three washes with RPMI, 2% FBS, the target cells were incubated with the effector cells at the indicated ratios for 4 hours. 100 ul of supernatant from each well was collected and the 5 lCr release was measured by a compugamma computer (LKB Instruments). The specific 5 lCr release was calculated as follows: [(experimental - media control) / (total -media control)] x 100%. The total release was obtained by lysis of the cells with a 5% Triton-X 100 (BDH) solution. 2.2e Peptide Transfer Assay Virus specific CTL effectors were generated as stated in 2.2c, with the LN being used on day three and the splenocytes on day six. VV infection of cold targets were as follows: trypsinized VV-NP, VV-pJS5, or VV-ESNP, along with 0.5 ml RPMI 2% HI FBS were added (MOI = 10) to cold targets that had been washed with PBS. They were incubated for 1.5 hours before adding 3 ml RPMI medium, 10 % HI FBS, 20 mM Hepes. The inhibitors brefeldin a (BFA) (10 p-g/ml), emetine (10-50 uM), and 31 azide (50 mM) were added as required for the times indicated, then the cells were incubated a further 3 hours before removing the cells with versene and 0.05 % Trypsin. They were washed two times, and then resuspended in CTL medium before addition to the CTL assay. RMA cells to be used as targets cells were radiolabeled with 51Cr for 1 hour at 37 °C and subsequently washed. 5 lCr was used at 100 uCi per 106 cells. Cold targets were diluted two or threefold as indicated, starting with 2 x 105 cells per well at a 20:1 ratio of cold/hot targets. Each well received 106 effector CTL and 104 radiolabeled targets (100:1 ratio effector/ hot targets). The CTL assay was then carried out as described in section 2.2d. 2.2f Limiting Dilution Analysis C57B1/6 mice were injected ip. with 106 pfu of VV-NP with or without 5 X 106 pfu VV-hTAP12. Six days later, spleens were removed and homogenized to yield a single cell suspension. A serial dilution of the single cell suspension was carried out and the cells were added, in replicates of 12, to a 96-well U bottomed plate along with 105 feeders plus 3000 stimulators per well. The feeders were naive, y-irradiated (5000 Rads) C57B1/6 splenocytes. The stimulators were VSV N peptide pulsed, y-irradiated (10,000 Rads) RMA cells. The cells were grown in RPMI complete medium (see section 2.2c) plus IL-2 (20 U/ml) containing rat T Stim (#40115, VWR) with 0.1 M a-methyl-D-mannoside. The cells were incubated for 6 days before performing a standard 4 hour 5 ,Cr release CTL assay (section 2.2d) where 100 pi of supernatant in each well was replaced with 100 pi of VSV N52-59 pulsed hot RMA targets (4000/well). A well 32 was considered positive if it exceeded the average plus three times the standard deviation of all negative controls wells (feeders and stimulator cells only). 2.2g Peptides The synthetic peptide representing the immunodominant epitope of the VSV N protein (amino acids 52-59: RGYVYQGL) was purchased from the University of Victoria Peptide Synthesis Facility (Victoria, B.C., Canada). The peptide was resuspended to lmM in PBS and stored in aliquots at -20 °C. 2.2h Animals The mouse strains C57BL/6 (H-2b), and Balb/C (H-2d), were obtained from Jackson Laboratories but housed and bred by Willem Schoorl at the Biotechnology Breeding Facility (University of B.C.). The H-2b nude mice [B/6 Nu-M (C57B1/6 NTAC-NufDF)] were obtained from Taconic (Hanover, NY, USA) and kept in specific pathogen free incubators. The mice were maintained according to the guidelines of the Canadian Council on Animal Care. The mice used in the experiments were between 6 and 12 weeks of age and were sacrificed by COz asphyxiation. 2.3 Viral Techniques 2.3a Viruses The viruses used in this thesis and their source are listed in Table 2. VSV stocks were grown on Vero cells in DMEM medium containing 10% FBS, P/S, and aliquots of the culture supernatant kept at -80 °C. Vaccinia virus (VV) stocks were 33 grown on CV-1 cells for small stocks and Hela S3 cells for the larger stocks. CV-1 cells were used to titre the VV stocks. The infections of the cell lines by VV were generally carried out at a MOI of 5-10. The VV was first trypsinized with 0.1 volume of 2.5 mg/ml trypsin (1:250: Difco Laboratories Inc., Detroit, MI) at 37 °C for 30 minutes, vortexing every 10 minutes, to break up the aggregated VV before being diluted in a small volume of DMEM media containing 2% FBS. This viral inoculum was added to cells previously washed with PBS and allowed to incubate for 60 - 90 minutes at 37 °C in a humidified, 5% C02/95% air environment. Then, complete medium containing 7 -10 % HI FBS was added and the cells allowed to grow until needed for an assay, or until the culture demonstrated 95% cytopathicity. When VV infected cells were required for an assay the infection was generally performed for 4-24 hours before the cells were removed with 0.25% trypsin, or versene plus 0.05% trypsin. When making a VV stock, the cells were removed using a cell scraper, centrifuged at 314 x g and the pellet resuspended in either culture medium, or a hypotonic solution of 10 mM Tris - HC1 pH 9.0, ImM EDTA. The stock was then freeze-thawed three times at -80°C then 37°C to release the VV from the cells. VV stocks were stored in aliquots at -80°C or -135 °C. 2.3b Recombinant Vaccinia Virus Construction Recombinant VV was constructed by homologous recombination of the wild type VV WR strain by infecting CV-1 cells transfected with the plasmids pJS5, or pJS5-rTAPl, or pJS5-VSVNP. This was done by infecting CV-1 cells with trypsinized 34 Table 2: Viruses used in this thesis Virus Description Source VSV Vesicular Stomatitis Virus (Indiana strain) F. Tuffaro UBC, Vancouver, Canada vv wild type Vaccinia Virus (WR strain) S. Gillam, UBC, Vancouver, Canada VV-ESNP VV containing amino acids 52-59 of the VSV N protein with an NH2- terminal E3/19K leader sequence J. Yewdell, National Institute of Health, Bethesda, MD. USA. VV-hTAPl VV containing human TAP1 J. Yewdell, National Institute of Health, Bethesda, MD. USA. VV-hTAP2 VV containing human TAP2 J. Yewdell, National Institute of Health, Bethesda, MD. USA. VV-hTAP12 VV containing human TAP1 and 2 J. Yewdell, National Institute of Health, Bethesda, MD. USA. VV-NP VV continuing amino acids 52-59 of the VSV N protein This thesis VV-pJS5 VV containing the vector only This thesis VV-rTAPl VV containing rat TAP1 This thesis 35 VV at a MOI of 0.05 and incubating for 2 hours at 37°C/5% C0 2. 10 ug of plasmid DNA in 1 ml. transfection buffer (0.14 M NaCl, 5 mM KC1, 1 mM Na2HP04-2H20, 0.1% glucose, 20 mM Hepes, pH 7.05) was precipitated with 50pl 2.5 M CaCl2 for 30 minutes before being added to the infected CV-1 cells. After a 30 minute incubation DMEM containing 10 % HI FBS was added and the cells were incubated another 3-4 hours before replacing the medium. After 2 days of incubation the cells were harvested with a cell scraper and centrifuged at 1800 x g (Beckman GP tabletop centrifuge) for 10 minutes. The pellet was resuspended in 0.5 ml of DMEM with 2 % FBS. The transfected cells were freeze/thawed 3 times (-80°C/37°C) before selection for the recombinant VV (rVV). 2.3c Selection of the Recombinant Vaccinia Virus For selection of the rVV, various dilutions of trypsinized transfected cell lysates (see 2.3b) were added to BS-C-1 cells and incubated for 2 hours at 37°C/5%C02. Once the inoculum was removed, the cells were overlay ed with the selection medium; DMEM containing 5 % FBS, 1% Noble agar, 0.25 mg/ml Xanthine, 25 ug/ml mycophenolic acid, and 15 ug/ml Hypoxanthine. After 2 days incubation, the viral plaques were removed and added to 0.5 ml DMEM, 2% FBS. The plaques underwent 2 more rounds of plaque purification with viral amplification between each round. Viral amplification was performed by trypsinizing and adding 100-250 pi of the plaque to 2.5X105 BS-C-1 cells/well of a 6 well plate and incubating until there was a 95% cytopathic effect. The scraped off cells were centrifuged and resuspended in 500 pi medium with 2% FBS and freeze/thawed three times to release the virus. 36 2.3d Southern blotting rVV clones Southern blotting was performed on each plaque in order to determine which plaques contained the rVV. Utilizing a slot blot apparatus, lOOul of an amplified plaque was added to Amersham Hybond N+ nylon membrane which had been prewetted with 5X SSC (0.3 M NaCl, 0.03 M NaCitrate, pH 7.4). The DNA was fixed onto the membrane with a 5 minute incubation on 0.5 M NaOH-saturated 3 MM Whatman. After rinsing twice with 5X SSC, the blots were air dried before hybridization. Amersham's ECL hybridization and blocking reagents were used for the prehybridization and hybridization steps. Probes were labeled with Boehringer Mannheim's terminal transferase kit and 32P-dCTP (Amersham) before being added to the hybridization solution. After incubating the blots overnight at 42°C they were washed twice with 0.1% SDS/0.1X SSC, then exposed to film (Kodak XAR). The probes used to verify the rVV were as follows: VV-NP plaques were verified using the 40mer oligonucleotide named (1) in 2.1b. The probe for VV-pJS5 was the 1704 bp gpt fragment from the pJS5 plasmid. Finally, the rTAPl plaques were verified using a TAP1 oligonucleotide probe with the following sequence: 5'-GAGTGTCTCGGGAATGCTGC-3'. 2.3e Purification of W Stocks Crude cell stocks were used for the infection of cells in culture however purified stocks of VV were used when injecting mice. To purify the VV, 3L batches of VV infected Hela S3 cultures were used. The VV was released from the cells by homogenization with a Dounce Homogenizer before centrifugation at 750 x g for 5 37 minutes at 4°C. The supernatant was trypsinized with 0.1 vol. of 2.5 mg/ml trypsin for 30 minutes at 37°C, then layered onto an equal volume of 36% sucrose in 10 mM Tris-HC1 pH 9.0. It was centrifuged for 80 minutes at 4°C at 25,000 x g and the pellet was then resuspended in 1 mM Tris-HCl pH 9.0. The pellet was trypsinized again before being layered onto a 24 - 40 % continuous sucrose gradient and centrifuged for 45 minutes, at 4°C at 18,750 x g. The milky band was collected and saved while the pellet was trypsinized and repurified on another sucrose gradient. All of the bands collected were pelleted by diluting with 2 volumes of 1 mM Tris-HCl pH 9.0 and centrifuging for 60 minutes at 4°C at 25,000 x g. The viral pellet was resuspended in 1 mM Tris-HCl pH 9.0, and 0.5 ml aliquots were stored at -80°C or -135 °C. 2.3f Titration of Viruses VV was titrated in CV-1 cells in a 6 well plate by first trypsinizing the virus, then adding 0.5 ml of the diluted virus (in DMEM, 2% FBS) to a well of PBS washed cells. After a one hour incubation in a 37°C humidified incubator, 3 ml of DMEM, 10% FBS was added and the cells with the virus were incubated for a further 2 days. To determine the plaque forming units per ml (pfu/ml) the cells were stained by incubating them for 5 minutes in 0.5 ml of 0.1 % crystal violet in 20% ethanol and the plaques counted. For VSV, the tissue culture infectious dose affecting 50% of the culture (TCID50) was used to determine viral titer as follows. Vero cells in 96-well plates were treated with various dilutions (102 - 10'4) of VSV. After 5 days incubation, the 38 individual wells showing cytopathic effect were counted as positive and the final titers for TCID50 were calculated according to Karber method. 2.4 Statistics The statistics for the cancer studies were performed using the Kaplan-Meier log rank survival test. The computer software program JMP IN version 3.2.1 (SAS Institute Inc. © 1989-97, Duxbury Press, NC. USA), was used to do the computations. The data was considered statistically different if p< 0.05. 39 3. Construction of Recombinant Vaccinia Viruses 3.1 Introduction In this project the ability of TAP to enhance an immune response to both a cancer and virus was determined. Various cell lines, as well as viral vectors containing either a TAP gene, or a minigene for a viral peptide were required for the study. The cancer experiments used a TAP deficient murine cancer cell line CMT.64. Also, cell lines derived from the transfection of CMT.64 with rat TAPs were obtained from G. Reid and R. Gabathuler (89,120). The stable CMT.64 transfectants contained cDNAs for rat TAP1 (CMT1.4, CMT1.10), rTAP2 (CMT2.1, CMT2.10), both rTAPl and rTAP2 (CMT12.12, CMT12.21), or the vector only (CMT.neo). The CMT.64 cell line and it's TAP derivatives were used to determine whether re-expression of TAP in CMT.64 limits the emergence of tumours, and malignancy related fatalities in vivo. However, in order to examine whether TAP could be used as a therapy, a viral vector containing rat TAP1 was required. Vaccinia Virus (VV) containing the rTAPl gene (VV-rTAPl) was constructed for this thesis. The experiments for increasing an immune response to a virus utilized VV carrying the human TAP molecules (VV-hTAPl, VV-hTAP2, VV-hTAP12) or a minigene encoding a viral peptide from the VSV N protein (VV-NP). The VV-hTAP constructs obtained from J. Yewdell (125) were co-injected with VV-NP into mice to determine whether the addition of TAP would increase the immune response to the viral peptide. The peptide chosen for the model antigen was amino acids 52-59 of the 40 VSV N protein. The VSV N peptide was chosen because it contains a cytotoxic epitope known to bind strongly to H-2K" and elicit a VSV specific CD8+ CTL response (126). This chapter demonstrates how the VV constructs VV-rTAPl and VV-NP were made, and confirms that they were functional. 3.1a Vaccinia Viruses as Vectors MHC class I molecules predominantly present peptides derived from proteins synthesized in the cell. Studies on antigen presentation by MHC class I have utilized many methods of targeting the antigens to MHC class I processing pathway. These strategies include adding lipophilic tails to peptides (127), transfecting cells with genes encoding proteins (128,129), and incubating cells with high concentrations of soluble peptides (130,131). However, none of these strategies are suitable for in vivo systems. One very popular method for endogenous expression of foreign proteins/peptides in animals is the use of viruses carrying the desired gene. This approach permits the appropriate expression of eukaryotic proteins, including post-translational modifications, correct folding and membrane insertion (132). Since some viruses, such as Vaccinia Virus (VV) undergo replication and transcription in the cytoplasm they are suitable vectors for the introduction of genes encoding protein antigens (133). Vaccinia Virus has been used as a viral vector for many years, not only for in vitro studies to introduce genes into the cell's normal endogenous protein processing pathways, but also for in vivo studies of gene therapy and for vaccination protocols (132,134). VV is a commonly used viral vector since it has been well studied and has 41 some very desirable properties such as its ability to accept large amounts of foreign DNA, its cytoplasmic transcription of proteins, its high protein expression levels, and its ability to infect a very broad host range (133). In this project VV was used both for in vivo and in vitro studies, as a vector for the expression of genes encoding the TAP proteins and the cytotoxic epitope of VSV N. 3.2 Results 3.2a Construction of VV-rTAPl and W-NP Recombinant VVs containing either rTAPl (VV-rTAPl) or the VSV N cytotoxic epitope (VV-NP) were constructed so that the VV would act as a vector for the introduction of the TAP and VSV N peptide genes into a mouse. The construction of VV-rTAPl (figure 4) involved the removal of the complete cDNA for rat TAP1 from a mammalian expression vector and then directly cloning the rTAPl gene into the VV shuttle vector, pJS5, creating pJS5-rTAPl. The rTAPl cDNA is the same as the one used to reconstitute the CMT.64 cell line to make CMT1.4, CMT1.10, CMT12.12, and CMT12.21 so that the same rTAPl will be used for the VV-rTAPl, and CMT experiments (89). A subunit vaccine was required in order to investigate whether the presence of TAP could enhance antigen presentation in vivo, so a model subunit vaccine (VV-NP) was created using VV as a carrier, and the immunodominant cytotoxic epitope (amino acids 52-59) of the VSV N peptide. The VSV N peptide was chosen because it is known to bind to H-2K" and elicit a specific CD8+ CTL response (126,135,136). A 42 Figure 4: Construction of pJS5-rTAPl The full length cDNA of rTAPl was isolated from the vector prip Apr-1 neo by a partial digestion with the enzyme EcoRI. The 2,500 bp fragment was purified from a 0.8% agarose gel and ligated into an EcoRI digested and dephosphorylated pJS5 vector to give pJS5-rTAPl. MCS refers to the multiple cloning site which contains many enzyme restriction sites. 43 VSVNP 5' C T A G C T C A G A G G C C T T G G T A G A C A T A T C C T C T C A T G A G C T 3 ' 3' G A G T C T C C G G A A C C A T C T G T A T A G G A G A G T A C 5 ' Figure 5: Construction of pJS5-VSVNP The minigene for the VSV N52-59 amino acids was made by annealing two complementary oligonucleotides coding for a methionine start site, the eight amino acid VSV cytotoxic epitope, and a stop codon. The double stranded minigene contains Nhel and SacI overhangs for direct ligation into the Nhel and SacI digested pJS5 plasmid, to give pJS5-VSVNP. MCS refers to the multiple cloning site which contains many enzyme restriction sites. 44 minigene encoding the cytotoxic epitope of the VSV N protein, amino acids 52-59, was created using oligonucleotides which were inserted into the pJS5 plasmid (figure 5) to create pJS5-VSVNP. The minigene included a methionine start site in front of the 8 amino acid coding sequence, as well as a translational stop codon at the end. DNA sequencing verified that the correct sequence and orientation of the minigene in pJS5 were correct. Once the pJS5-rTAPl and pJS5-VSVNP plasmids were constructed they were used to create the rVV containing the rTAPl (VV-rTAPl), or VSV N52-59 (VV-NP). A rVV vector only control (VV-pJS5) was also constructed. The rVV were made by transfecting the plasmids into the VV infected B-SC-1 cell line where the wild type VV and transfected plasmids underwent homologous recombination at the thymidine kinase (tk) gene of VV. Once the rVV were isolated by selection with XMH (see 2.3c), and recombination was verified by southern blotting, large rVV cultures were grown for later purification of the rVV on sucrose gradients. Purification of the rVV from the cellular debris was considered essential in order to eliminate any immunological responses by the mice to cellular material from Hela, CV-1, or BS-C-1 cells. Crude cell viral lysates were used for infecting cells in vitro. 3.2b Functional Analysis of VV-NP, VV-TAP1, and W-pJS5 Once the VV-rTAPl was constructed a western blot analysis was performed to confirm that the virus was functional and actively produced rTAPl. The production of rTAPl in VV-rTAPl infected cells was detected by western blot analysis as seen in 45 figure 6. After a two day viral infection the rTAPl protein was seen in lane 3 in the VV-rTAPl infected CV-1 lysate and is expressed with the expected size of approximately 70 kDa. In lanes 1 and 2 both the noninfected CV-1 lysate and the WT VV lysate showed no rTAPl production. Since the TAP complex is responsible for transporting peptides across the ER lumen for binding to MHC class I the functional assay for the rTAPl involves the restoration of antigen presentation in a TAP deficient cell. In order to determine this, a CTL assay specific for the VSV N52-59 cytotoxic epitope was used to verify that the rTAPl produced by VV-rTAPl was functional (Figure 7). The TAP complex requires both TAP1 and TAP2 to transport peptides into the ER and the target cell line, T2Kb, used in this assay is deficient in both TAP molecules. The TAP deficiency causes low MHC class I levels on its cell surface, which results in poor recognition by CTL. However, reconstitution of T2Kb with both TAP molecules restores peptide transport into the ER as well as cell surface presentation of peptide by MHC class I (137). To provide a complementary TAP2 molecule for VV-rTAPl, a rVV containing the human TAP2 molecule (VV-hTAP2) was used. The reporter peptide used in this experiment came from VV-NP since rTAPl has been shown to transport the cytotoxic epitope of the VSV N peptide (89,120). This CTL assay showed that T2Kb does not present peptide when there are no TAP molecules present or when only TAP1 or TAP2 is present. The vector only control, VV-pJS5, also did not present the VSV N peptide nor did the VV infection interfere with the cell's ability to transport peptides into the ER. However, when both TAP molecules were present as either the human or rat:human heterodimer, the VSV N peptide was transported into the ER and recognized by the 46 CV-1 CV-1 + WT VV CV-1 + VV-rTAPl KDa 97 68 rTAPl 43 -Figure 6: Expression of rTAPl in a W-rTAPl infection of CV-1 cells The expression of rTAPl was examined by western blot in W - r T A P l infected CV-1 cells. NP-40 lysates from 106 CV-1 cells, with or without a two day VV-rTAPl infection, were applied to a 12 % SDS-PAGE before transfer onto immobilon-P PVDF membrane. The membrane was probed with the rTAPl specific polyclonal antibody D90, followed by donkey anti-rabbit-biotin IgG. The rTAPl protein was detected by using BioRad ECL reagents and SA-HRPO, before exposing the membrane to film. 47 50 45 40 35 '55 _ i 30 o 25 >P o (D a 20 15 10 5 0 3 1 0.3 Effector:Target Ratio • T2Kb gW-NP+W-pJS5 • W-NP SW-NP+W-hTAP1 • W-NP+W-hTAP2 DflW-NP+W-rTAPI ^W-NP+W-rTAP1 +W-hTAP2 ^W-NP+W-hTAP1 +W-hTAP2 Figure 7: Determination of VV-NP expression and VV-TAP function A CTL assay was performed to determine the ability of VV-NP to produce the VSV N cytotoxic peptide as well as the ability of VV-TAP to produce a functional TAP which transports the peptide. The targets T2Kb were infected (MOI = 10) for 6 hours with either VV-NP, or VV-NP with various combinations of the VV-TAP. The presentation of peptide was determined indirectly by target cell lysis by VSV specific CTL in a 4 hour 5 lCr release assay. 48 VSV specific CTL. This assay not only suggested that the rTAPl in VV-rTAPl actively transported peptides into the ER but also that the minigene for VSV NP in VV-NP was translated, could bind to MHC I, and was recognized by VSV-specific CTL. The TAP molecule-encoding VV that were used in conjunction with the VV-NP for the immunization experiments were the VV-hTAPl, VV-hTAP2, VV-hTAP12 recombinant viruses obtained from J. Yewdell. Figure 7 confirms that the VSV specific CTL were able to recognize the VSV N peptide in T2Kb cells reconstituted with the human TAPs. This showed that the VV-hTAPs are functional only when present in combination and that they were able to transport the VSV N peptide. 3.3 Discussion VV has been used for the endogenous production of many proteins (138-142). In this project, VV was used as a vector to express the VSV N peptide and rTAPl. VV was chosen as the vector because it undergoes transcription and translation in the cytoplasm which ensures the protein will follow the cell's normal protein processing pathways. It was important to produce the VSV N peptide in the cytoplasm so that it would be available for transport by the TAP complex. The rVV containing the genes for the peptide and rTAPl were successfully made and were functional. In VV-rTAPl infected cells, the rTAPl produced was of the correct molecular weight and was able to act in conjunction with hTAP-2 for transport of the peptide VSV N52-59. In regard to VV-NP, the peptide was successfully made in the cell and presented by MHC class I on the cell surface for recognition by VSV specific CTL. Overall, the rVV constructs are 49 suitable for the endogenous production of the TAP protein and VSV N peptide. Moreover, the VV does not interfere with the normal MHC I antigen processing and presentation pathway. 50 4. Using TAP as an adjuvant 4.1 Introduction Adjuvants have been described as any agent that enhances the immune response to an antigen. Therefore, adjuvants have been used extensively in vaccines where a strong immune response against an antigen is required. In order to elicit a cytotoxic memory response to viruses the primary goal is to get the antigen presented by MHC class I on the cell surface. TAP is an integral component of the antigen processing and presentation pathway and the rate of transport of MHC classl:peptide to the cell's surface is dependent upon the efficiency of peptide transfer in the ER by TAP to 'empty' MHC class I (94,95). In this chapter TAP was added to a wild type cell to determine if it could increase antigen presentation and therefore enhance antigen specific cytotoxic responses. 4.1a Vaccine development The initial defense mechanism that occurs upon exposure to a pathogen for the first time is not always specific or rapid enough to protect against the lethal or debilitating diseases caused by organisms like the smallpox virus, or polio. It is in these cases that we need to acquire specific immunity in order to lessen the devastating effects that occur upon infection. Harnessing the immune system to protect against infectious disease agents is the goal of vaccination. For centuries the search for methods of providing immunity through vaccination has helped us eradicate smallpox and has provided a basis for understanding the immune system and how to elicit immunological memory against viruses and bacteria. 51 One of the earliest documented cases of providing immunity safely was when Jenner realized that milkmaids did not get smallpox. Therefore, he used the cowpox virus to successfully immunize people (143,144). As knowledge about what organisms were responsible for diseases increased, the various viruses and bacteria were isolated and used in vaccines. The vaccines at that time usually included either a closely-related but less virulent pathogen or an inactivated form of the whole pathogen (144). Examples of diseases in which live attenuated virus or bacterial vaccines were used successfully included measles, mumps, polio, and tuberculosis, as well as many others (1). There were problems with these early vaccines (145). The inactivation process did not always neutralize the organism and the vaccine sometimes caused the disease it was trying to prevent. Also, the inactivation process sometimes weakened the protective immune response which it was supposed to elicit thereby failing to induce long lasting immunity. Today research continues with the aim of making a vaccine that is safe, inexpensive, stable, and able to provide long lasting immunity (1). Due to the technological advances that have occurred in molecular biology and immunology, a new type of vaccine has emerged which uses only the most immunogenic pieces of a pathogen. Subsequent vaccines that have been developed include the Hepatitis B surface antigen and the pneumococcal capsular polysaccharides (146,147). These new subunit vaccines can provide protection against a specific pathogen and should theoretically be safer since it does not introduce the live organism that causes the disease. 52 4.1b Subunit vaccines Knowledge of how the immune system works has evoked many creative ideas on how to construct synthetic subunit vaccines (148). For the most part, protective immune responses were measured by the amount of circulating protective antibodies against a pathogen. Therefore, the early vaccine models used whole proteins since antibodies tend to bind to tertiary conformations of native or denatured proteins (reviewed in[149]). However, the antibody response is generally directed towards extracellular pathogens and their toxins. In the last decade or so, information from two aspects of the immune system has allowed us to go beyond invoking just a B cell response. One is the discovery that T lymphocytes could also protect against viral pathogens. The other is the knowledge that T cells respond to short peptides bound to MHC molecules on APC. This has led to the development of T cell vaccines (150). Depending on whether a CD8+ or CD4+ response is required, the size of the peptide fragment required for a T cell vaccine should range from 8-10 amino acids for MHC class I, or 10-34 amino acids for MHC class II. These sizes correspond to the size of peptide that fits into the MHC peptide binding grooves. Due to the different antigen processing pathways that MHC class I and II use, the methods for peptide delivery to evoke a CD4+ T cell response can be different than for a CD8+ response (150). In many viral infections it was found that the CD8+ cytotoxic response was most effective in eliminating the virus and providing protection, so for this reason peptides containing cytotoxic epitopes that bind to MHC class I are being investigated for possible use in vaccines (109,151-154). While MHC class II requires an exogenous delivery of peptides, MHC class I requires cytosolic peptide delivery. Since peptides 53 are transported poorly into cells, new methods of delivering immunogenic peptides have been developed such as adding lipophillic tails to peptides (127), incorporating the peptide into liposomes (155), ISCOMS (156-158), or safe viral vectors (132). Two of the most popular viral vectors that have been used to present foreign proteins are adenovirus (159), and VV (134). While adenovirus can provide a protective response at lower titres, it is more heat labile, has a narrower host range, and cannot tolerate as much DNA to be encorporated into the genome as VV (160,161). Despite the pros and cons of each virus, both have been effective in eliciting a protective immune response against the foreign protein they encoded (108). The use of VV as an immunization vehicle for humans has been questioned due to complications from vaccination such as post-vaccinal encephalomyelitis and other neurological defects (108). However, many safer non-replicating and highly attenuated VV strains have been created that are safer (162,163). When rVV containing rabies antigen is injected into dogs and cats, it protects against lethal viral challenge (164). The attenuated avipox virus ALVAC containing the human immunodeficiency virus type 1 envelope protein has been used to elicit specific immune responses in phase I clinical trials (134). 4.1c Adjuvants Another aspect in vaccine development is the incorporation of adjuvants in order to get rapid clonal expansion. Alum is currently the most widely used adjuvant but it has a number of drawbacks (165,166). Not all proteins can be adsorbed well onto alum, it elicits a poor T cell response and aluminum-related pathologies have been 54 reported (165). New adjuvants, created to increase immunological memory, have been designed in accordance with the immunogenic antigen used and the type of immune response that needs to be elicited (167). The list of adjuvants is long, so a short summary of the more common ones is listed in table 3 (reviewed in 168,169). With the use of adjuvants the immune response can be modulated for an MHC class I or II response. Adjuvants like immunostimulating complexes (ISCOMs), that are made of non-covalenty bound complexes of Quil A, cholesterol, and amphipathic antigen can stimulate a CD8+ CTL response (170). Similarly, the T cell costimulatory molecule B7 has been shown to enhance protection against poorly immunogenic tumours (171,172). In addition a wide variety of cytokines have been used to direct responses to either a CTL mechanism or T helper response. For example, interleukin-2 (IL-2) and IL-12 have been used to elicit a Thl response which is more conducive to cytotoxic mechanisms (173-177). One adjuvant that has been widely used in animals is Freund's complete adjuvant (FCA) which is an emulsion containing heat killed mycobaterium tuberculosis. Despite the strong antibody responses that FCA produces, it is too toxic be used in humans. However, derivatives of the minimal structure of the mycobaterium in FCA that is needed for adjuvanticity, N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), such as murabutide do not have any toxicity problems (168). Many more adjuvants are being developed and are improving vaccines against a wide variety of pathogens. One method of increasing the immune response to a 55 cytotoxic epitope will be examined in this work, supplying TAP along with the immunogenic peptide. Table 3: Summary of some common adjuvants Adjuvant Immunomodulation aluminum salts strong Th2, IgE water-in-oil or oil-in-water emulsions weak Thl and Th2 ISCOMs strong Thl and Th2 lipid A (MPL) strong Thl cytokines Thl => IFN-y, IL-2JL-12 Th2 => IL-4 MDP strong Thl (lipophilic) or Th2 (hydrophilic) Freund's adjuvant strong Th2 saponins strong Thl, Th2 4.Id TAP as an adjuvant The TAP complex is responsible for maintaining a supply of peptides to the MHC class I and it has been suggested that the supply of peptides may be a limiting factor in the number of stable MHC class I on the cell surface (94,95). TAP retains 56 'empty' MHC class I in the ER until it binds peptides. If increasing the expression of TAP to a cell could increase the number of MHC class I molecules on the cell surface then perhaps the inclusion of genes encoding TAP with a gene encoding a cytotoxic epitope in a VV vector could increase the specific antigen presentation, this may result in a greater immune recognition of the displayed antigen. In this chapter, the efficacy of using the TAP gene as an adjuvant in a viral subunit vaccine has been investigated to see if it can enhance a CTL mediated immune response against a cytotoxic peptide. 4.2 Results 4.2a VV-NP as a model subunit vaccine In order to test the effect TAP would have as an adjuvant, a model subunit vaccine VV-NP was constructed (see chapter 3). Minigenes in a VV vector have been used to deliver cytotoxic epitopes to MHC class I. Minigenes are very efficient in providing peptides in the cytoplasm. It was determined that a rVV containing a minigene encoding an 8 amino acid peptide from an influenza protein could produce 8,000 peptides per hour in comparison to the entire influenza protein which provides antigenic peptides three to four orders of magnitude less efficiently (179). In this project a minigene in rVV provides an effective way to use the cytotoxic epitope of the VSV N protein (VSV-NP) as a reporter molecule to determine the level of immune responsiveness in mice (180-182). Before TAP can be used along with the subunit vaccine, VV-NP had to be tested in vivo to see if it could elicit an anti-VSV response. As seen in the CTL assay in figure 8, the splenocytes from mice injected with VV-NP 5 7 A 10 3 1 0.3 0.1 Effector:Target B 100 .. 80 -m '55 60 -> _i 40 -°^ 20 ( 10 3 Effector:Target -•—VSV -•-W-NP-6 - A — W-pJS5 -e-PBS Figure 8: Immune response to varying W-NP dosage Splenocytes from immunized C57B1/6 mice were tested for their ability to recognize VSV infected targets in a 4 hour 51Cr release CTL assay. The mice were injected ip.with either VSV (3X107 TCID50), or VV-NP at 103 (3), 104 (4), 105 (5), 106 (6) pfu, or VV-pJS5 (106 pfu), or PBS. The splenocytes were tested for their ability to recognize either A) RMA targets infected with VSV (MOI = 10 for 8 hours) or B) uninfected RMA targets. 58 were able to lyse RMA targets infected with VSV but not uninfected RMA targets. The minimum amount of VV-NP required to elicit an immune response in C57BL/6 mice was 104 plaque forming units (pfu) and the maximum immune response was achieved at 105 - 106 pfu. At a higher titer of 108 pfu (107 and 108 not shown) the response decreased and VV levels above 108 pfu were lethal. There does not appear to be any significant difference in the response elicited by doses of VV-NP between 105 and 107 pfu, so the median dose of 106 pfu was chosen for the remaining assays. The splenocytes in the mice injected with VV-NP in figure 8 included cytotoxic lymphocytes which were specific for VSV. It was also determined in a CTL assay with VSV N52-59 pulsed targets, that the immune response elicited in mice injected with VV-NP included CTLs that were specific for the VSV N52-59 cytotoxic epitope (figure 9). The mice injected with the control vector VV-pJS5 did not have VSV specific lymphocytes as they gave the same low response seen in mice injected with PBS. Splenocytes from the VV-NP primed mice were also able to recognize VV infected RMA targets but to a much lesser degree than they recognized the VSV N peptide epitope. 4.2b TAP enhances a specific immune response In a VSV-specific CTL assay shown previously in figure 7 (section 3.2b) the VSV N peptide is a TAP dependent peptide and therefore provides a good readout for TAP transport. The assay also demonstrated that the peptide could be transported by the human TAP complex. The next step was to determine whether including the TAP gene along with the VV-NP in a VV vector would increase the number of VSV N 59 A 100 _ 30 10 3 1 EffectonTarget Figure 9: Specificity of splenocytes from VV-NP immunized mice. Splenocytes from immunized C57B1/6 mice were tested for their ability to recognize targets in a 4 hour 5 lCr release assay. The mice were injected ip. with either VSV (3X107 TCID50), or VV-NP (106 pfu), or VV-pJS5 (106 pfu), or PBS. They were tested for their ability to recognize either A) RMA targets pulsed for one hour with VSV N peptide or B) RMA targets infected with VV-pJS5 (MOI = 10 for 8 hours). 60 peptide-specific cytotoxic lymphocytes in the spleen. A CTL assay was used to measure the response and an increase in lysis of the VSV infected RMA targets indicated an increase in the number of VSV specific CTL (figure 10). I found that the in vivo immune response to VV-NP was amplified when the mice were simultaneously injected with the VV-hTAPs. The largest increase in response over VV-NP alone was seen with VV-hTAP12, and to a lesser degree with VV-hTAPl and VV-hTAP2. This suggests that the addition of a TAP gene enhanced the VSV NP-specific CTL response. It also demonstrated that including both TAP1 and TAP2 genes was more effective than using either TAP1 or TAP2 alone. A preliminary limiting dilution analysis (LDA) was performed to confirm that mice which received VV-hTAPl2 and VV-NP contained more VSV specific CTL. The LDA data suggests that mice which received VV-NP had 1 VSV specific pCTL for every 78,000 splenocytes, whereas mice which also received VV-hTAP12 had 1 pCTL for every 16,000 splenocytes (figure 10b). The addition of TAP increased the number of VSV specific CTL approximately 5 fold. To determine if VV-TAP could be effective when used at low doses mice were immunized with VV-NP (103 pfu) and VV-hTAP12 (5X103 pfu) at doses which provided the minimum immune response in mice (figure 11a). Mice received 1000 times less VV-NP and VV-hTAPl2 (ratio of 1:5) than was used before. The level of the immune response was assessed using a CTL assay with peptide-pulsed RMA as targets. In comparison to the VV-NP only response, the addition of VV-TAP 12 appeared to abrogate the VSV N response. This suggests that the response against VV prevents priming against the VSV N peptide carried by VV. This was confirmed by the 61 A •W-NP 10 3 S 40 90 B— VV-NP+ VV-hTAP1 A—VV-NP + W -hTAP2 e—W-NP+ VV-hTAP12 <e-W-pJS5 30 10 Effector:Target B VV-NP VV-NP + VV-hTAP12 VSV pCTL 1/78,000 1/16,000 Figure 10: Effect of TAP on W-NP immunization A) Splenocytes from C57B1/6 mice immunized ip. with VV-NP (106 pfu) with or without VV-hTAP (5X106 pfu) were tested for their ability to recognize VSV infected RMA targets (MOI = 10 for 9 hours) in a 4 hour 5 lCr release assay. B) The frequency of VSV specific CTL in splenocytes from mice injected with VV-NP alone or with VV-hTAP12 were determined in a limiting dilution analysis. 62 A Figure 11: Effect of TAP on a low dose immunization Splenocytes from C57B1/6 mice were tested against VSV NP (1 uM) pulsed RMA targets in a standard 4 hour 51Cr release assay. The mice in A)were immunized with VV-NP (103 pfu) alone, or along with either VV-hTAP12 (5 X 103 pfu) or VV-pJS5 (5 X 103 pfu). The mice in B) were immunized with VSV (2.7 X 103 TCID50) alone or with VV-hTAP12 ( 1.35 X 104 pfu). 63 fact that adding the VV-pJS5 vector only control along with VV-NP also reduced the response to the VSV N peptide. To eliminate this problem with the VV carrier, the same experiment was attempted using a low dose of VSV (2.1X103 TCID50) along with 1.35X104 pfu VV-hTAP12 (figure lib). The dose of VSV used to give the minimum immune response was approximately 10,000 times less than the usual dose used for VSV immunization. It appears that immunizing with VV-hTAP12 and VSV resulted in a large increase in immune responsiveness against VSV. This suggests that TAP could be a suitable candidate for increasing an immune response to low doses of antigen. The initial hypothesis as to how TAP could increase an immune response was that the supply of peptides available to the MHC class I cycling to the surface was limiting (94,95). Therefore, the amount of antigen displayed on the cell surface could potentially be increased if the level of cellular TAP was also increased. The CTL assay provides a good readout of the level of antigen presentation as demonstrated in dose-response experiments of peptide pulsed targets where greater specific lysis is seen with increasing peptide concentrations (89,183). An in vitro assay was used in which the targets where infected with VSV with or without VV-TAP. In this assay VSV specific CTLs were used to determine whether increasing TAP expression above normal levels would increase the amount of VSV N peptide presented in RMA targets (figure 12). I found that the addition of VV-hTAPs, as either TAP1 or TAP2 individually or together, did not have an effect on the amount of peptide presented as there was no significant difference in the lysis of the targets. Similar results were obtained when the targets where infected with VV-NP instead of VSV (data not shown). This suggests that 64 Figure 12: TAP does not augment VSV antigen presentation in RMA cells in vitro The ability of TAP to increase antigen expression on the surface of RMA cells was determined by a 4 hour 5 lCr release assay. The effectors were VSV specific CTL. The targets were RMA cells infected at an MOI = 10 for 5 hours with VSV plus or minus VV-hTAP. 65 the increase in the immune response seen in the in vivo immunization and low dose experiments (figures 10 and 11) was probably not due to an increase in peptide presentation by MHC class I on the surface of the cell. 4.3 Discussion Adjuvants are described as any substance that enhances the immune response to an antigen. We have shown, using the subunit vaccine model, that TAP does increase the immune response to the VSV N peptide. The peptide used was a cytotoxic epitope which binds to H-2Kb, therefore we were able to show that the killing was mediated by CD8+ CTL. Also, because TAP is so effective, over 10,000 fold less virus could be used. When designing subunit vaccines one wants to reduce the amount of innoculum used for vaccination. This is important not only for keeping costs and shipping down when making and distributing large amounts of a vaccine but is also important when larger doses of a weakly immunogenic peptide are required or when multiple disparate antigens are used (1,9). The use of TAP could help increase the effectiveness of limited quantities of peptide. This would serve two purposes. It would keep the inoculum down to a very small volume therefore making it cost effective. In addition it would increase the response to poorly immunogenic peptides. Providing VV-TAP on a separate VV was a problem in this study as it was necessary to infect with both VV-NP and VV-TAP in order to get an increase in peptide presentation. This meant using a ratio of 1:5 of VV-NP to VV-TAP in order to increase the chances that a cell which produced the VSV N peptide was also infected 66 with VV-hTAP. In low dose immunizations this resulted in the bulk of the response to be against VV instead of VSV NP. However, this problem could be overcome in the future by supplying the TAP molecule and cytotoxic epitope in the same VV vector. Overall, it appears that TAP could be used as an adjuvant in peptide vaccines but it does not have to be restricted to viral vectors. It could also be injected in other forms such as in DNA plasmids attached to gold particles or any other system which inserts the TAP complex directly into the cell's protein processing pathway (148). VV is one of the most utilized viral vectors at this time because it undergoes replication and transcription in the cytoplasm and can incorporate large amounts of foreign DNA into its genome. More attenuated forms of the VV are being made to counteract the potential hazards of immunizing with the live virus. Possible alternatives to VV are the highly attenuated NYVAC strain of VV (161), and the avipox virus, ALVAC (162) which does not replicate in nonavian cells. 67 5. TAP is involved in surrogate antigen presentation 5.1 Introduction CTL recognize antigenic peptides bound to MHC class I molecules that are expressed on the surface of virally infected cells. It has been well established that the supply of peptides for MHC class I assembly comes primarily from peptides degraded in the cytosol by the proteasome which are then transported into the ER by TAP. It has been proposed that ER lumenal peptides that cannot bind to MHC class I or are in excess of MHC class I are rapidly degraded (109,110). Alternatively, these surplus peptides may bind, independently of MHC class I to the ER resident heat shock protein (HSP) gp96 (111). Evidence that ER peptides have an alternative to binding MHC class I was demonstrated when the HSP purified from a cell was associated with a broad range of cellular peptides. Furthermore, many of these peptides were determined to be cytotoxic peptides as immunization with the peptide bound gp96 were able to induce specific CTL responses against a variety of intracellular antigens (184,185). Other studies looking at TAP transport in microsomes have shown that peptide transport is reversible in an ATP and temperature dependent manner (86,112). This suggests that peptides not bound to MHC class I in the ER lumen may be transported back into the cytoplasm by an efflux mechanism which has yet to be elucidated. Since there are peptides in the ER that are not bound to MHC class I, there may also be an additional fate for these peptides other than the ones described to date. It is possible that the peptides could be secreted via the constitutive secretory pathway. This pathway is the normal route used by proteins that are secreted from the cell and 68 involves the transport out of the cell through the ER, Golgi, trans Golgi reticulum, and secretory vesicles to the plasma membrane (186). The constitutive secretory pathway normally involves the translocation of proteins into the ER lumen via the translocon (187). Proteins targeted for translocation into the ER contain a signal sequence, or signal anchor sequence which are recognized by the signal recognition particle (SRP). The complex then interacts with the SRP receptor on the ER membrane at the translocon. The translocon is a multimeric protein complex comprised of a minimum of six polypeptides (sec61ct,sec61p\sec61y, two SRP receptors, and TRAM) which were identified in crosslinking experiments (188-191). Additional components are believed to optimize the translocons activity. Some of these components include the five polypeptide signal peptidase complex and the three polypeptide oligosaccharyltransferase complex which are involved in signal cleavage and glycosylation, respectively (192). Although the translocon is thought of as the transporter involved in protein secretion, it is clear that cytosolically derived peptides are transported into the ER by TAP. In this chapter, the role of TAP in the secretion of peptides and the effect of this secretion on CTL mediated killing were examined. 5.2 Results To examine if TAP plays a role in the secretion of antigenic peptides out of the cell, a modified CTL assay (peptide transfer assay, see 2.2e) was used to measure whether virally infected cells could transfer sensitizing peptide to uninfected neighbouring cells (183). In the peptide transfer assay the CTL are VSV specific but the targets are a mixture of unlabelled VV-NP infected cells, and chromium (5lCr) 69 loaded uninfected RMA targets. The infected cells used in this assay were either a TAP deficient CMT.64 cell line, or the TAP1 and 2 expressing CMT12.12 and L cells. The transfer of peptide from virally infected unlabelled cells to the radiolabeled RMA targets was measured by the release of 5 1Cr when the RMA cells are lysed by the VSV specific CTL. The peptide transfer assay showed significant lysis of the uninfected RMA cells when incubated with VV-NP infected L cells that express TAP1 and 2, suggesting that sensitizing peptides were transferred and that the phenomenon was titratable (figure 13a). The L cells (H-2Kk) used in this assay are not recognized by the VSV specific H-2Kb restricted CTL. This indicated that the peptides that bound to radiolabeled RMA targets did not arise from the lysis of the infected L cells by the CTL. It also suggested that the peptides were not being shed from the cell surface MHC class I of the L cells, and that bystander killing of the RMA cells was negligible. Since the peptides do not bind to the L cells, then the interaction with and transport by MHC class I molecules is not a prerequisite for peptide transfer. This was confirmed by Gabathuler et al when RLE (H-2k) cells, which do not readily transport heavy chain MHC class I to the cell surface because they lack p2m, were able to efficiently transfer peptide to RMA cells (data not shown, see 183). Further work on the peptide transport assay by Gabathuler et al demonstrated that viral mediated cell lysis does not mediate peptide transfer and that the level of lysis represents exposure of the uninfected cells to approximately 5-50 nM of VSV N peptide (data not shown, 183). Overall, it appears that peptides were secreted by infected cells and sensitized neighbouring RMA cells to CTL lysis. 70 A 35 HL+W-NP H L+VV-ESNP • CMT.64 + W-NP a CMT.64 + W-ESNP 20 6.7 Cold:Hot Ratio Figure 13: VSV NP can be transferred to and sensitize uninfected RMA cells in a TAP-dependent manner A peptide transfer assay was used to detect the ability of infected cells to transfer VSV N peptide to neighbouring uninfected cells. A) The cold L, CMT.64, and CMT12.12 cells were infected with VV-NP or VV-pJS5 (MOI = 10 for 6 hours.) Or B) the cold L, or CMT.64 cells were infected with either VV-NP or VV-ESNP (MOI=10 for 6 hours). In both figures the hot targets were uninfected RMA (H-2b) cells loaded with 5 1Cr. The effectors were VSV specific CTL. The degree of lysis of the hot RMA targets was measured after 4 hours and reflected the amount of transfer of VSV N peptide from the infected cold targets to the hot RMA targets. 7 1 To investigate whether the TAP complex was important in the secretion of the peptides, cells deficient in TAP were used as the peptide donors in the peptide transfer assay (figure 13a). CMT.64 cells which are deficient in TAP 1 and 2, were unable to transfer peptides whereas TAP12 containing L cells, and CMT12.12 cells were able to transfer peptide. This demonstrates a dependency on the expression of the TAP transporters for the exchange of peptides suggesting the peptides must be transported into the ER lumen in order to be transferred to neighbouring cells. As further confirmation that the VSV N peptide secretion was TAP dependent, a VV containing an ER signal sequence in front of the VSV N52-59 coding sequence (VV-ESNP) was used to infect the donor cells (figure 13b). The signal sequence allows the peptide to be transported by the translocon into the ER and eliminates the need for translocation by TAP. In this case cells infected with VV-ESNP showed peptide transfer in both the TAP deficient CMT.64 cells as well as the TAP containing L cells. Only the TAP expressing L cells infected with VV-NP were able to transfer peptide whereas the TAP deficient CMT.64 cell line did not. The VV-NP had a slightly higher level of lysis than the VV-ESNP infected L cells. This may be due to the fact that the signal sequence requires additional processing. Alternatively, it could be due to heterogeneous expression levels between the two recombinant viruses. The peptide can bind the MHC class I on the radiolabeled target cells presumably due to the trimming of the signal sequence in the ER which would release the VSV N peptide. These results confirm that the secretory pathway was utilized and that transfer was mediated by the TAP complex. 72 The role of the secretory pathway in peptide transfer was further studied using BFA as a protein secretion inhibitor. Infected cells were treated with BFA for the stated time, before addition to the peptide transfer assay. As seen in figure 14a, there was peptide transfer in untreated CMT12.12 and L cells, however when treated with BFA peptide transfer was inhibited. In the case of the cells that were treated with BFA and also infected with the TAP-independent VV-ESNP we saw that peptide transfer was also inhibited in comparison to non-BFA treated samples (figure 14b). These data suggests that peptide transfer can occur with peptides that enter the ER via the TAP complex as well as from the translocon. Incubation of infected cells at low temperature as well as the use of other cell inhibitors also had an effect on the peptide transfer assay. Emetine, an inhibitor of protein synthesis (193) and azide which blocks respiratory metabolism, also prevented the transfer of peptides from infected L cells (figure 15). A reduction in temperature has been shown to block protein secretion (194). In the peptide transfer assay, a decrease in lysis was seen when there was a drop in temperature from 37°C to 21°C for 3 hours before the start of the CTL assay. However, the blockage was not complete. This was most likely due to a release of the temperature block when the donor cells were added to the CTL assay. Together these inhibitors demonstrate that protein synthesis and energy metabolism are required. The results showed that an active secretory pathway, including the transport of peptides by the TAP complex, is required to transfer antigenic peptides from a virally infected cell to a neighbouring uninfected cell. 73 A T T M=wM 20 6.7 2.2 Cold:Hot Ratio HL+W-NP IL+W-NP+BFA • CMT.64+W-NP gCMT.64+W-NP + BFA ^CMT12.12+W-NP ^CMT12.12+W-NP+BFA B 20 15 % 10 6.7 2.2 Cold:Hot Ratio B L+W-ESNP @L+W-ESNP+BFA • CMT.64+W-ESNP g CMT.64+W-ESNP+BFA CMT12.12+W-ESNP CMT12.12+W-ESNP+BFA Figure 14: BFA inhibits peptide transfer The transfer of peptide from infected cold targets was inhibited by BFA in a peptide transfer assay. L cells, CMT.64, or CMT12.12 cells were infected with either A) VV-NP or B) VV-ESNP at an MOI = 10 for 6 hours. BFA (10 ug/ml) was added 2 hours after the start of the infection. Lysis of hot RMA targets by VSV specific CTL was measured in a 4 hour 5 lCr release assay. 74 A BL+W-NP • L+W-NP+ Em gL+W-NP + 21C 6.7 2.23 0.74 Cold .Hot Ratio B BL+W-NP • L+VV-NP + azide 5 2.5 1.25 Cold:Hot Ratio Figure 15: Emitine and azide inhibit peptide transfer The transfer of peptide from infected cold targets was inhibited by A) emitine (10 uM), low temperature, and B) azide (50mM), in a peptide transfer assay. L cells were infected with VV-NP at an MOI= 10 for 6 hours. Addition of emitine and azide, as well as the shift to 21°C, started three hours after the start of the viral infection. Lysis of hot RMA targets by VSV specific CTL was measured in a 4 hour 5 l Cr release assay. 75 5.3 Discussion In the peptide transfer assay it was determined that the endogenously produced VSV N peptide was transferred to neighbouring cells in a TAP dependent manner and did not involve the translocon. The confines for this transport mechanism will be the size and composition of the peptides that can be transported by the TAP molecules. The specificities of the TAP molecules from different species, and different TAP alleles within a species may alter the type of peptides delivered to the ER (92,101,112). The transferred peptides did not originate from surface MHC since peptide pulsed RMA cells were unable to sensitize neighbouring targets to lysis (183). This assay does not identify the mechanism of peptide secretion or whether a carrier may be involved. However, secreted 'free' peptides could be transferred to a neighbouring cell and displace peptide from MHC class I on the cell surface. Alternatively, the peptide could be bound to a carrier molecule such as a HSP, and internalized by neighbouring cells. Once inside the cell the peptide could enter the class I biosynthetic pathway and bind to MHC class I trafficking to the cell surface. Further studies are required to determine the peptide transfer mechanism. Secretion appears to be independent of binding to MHC class I which could be due to an excess of peptide or because the MHC which binds the peptide is absent. Since viral proteins tend to be made in large quantities, the peptides produced may be in excess as in the experiments done here. In this case the limitation in the number of TAP dependent viral peptides secreted will depend on the number of TAP complexes and the speed with which they transport peptides into the ER. The secreted peptides would then bind to neighbouring cells making them prone to lysis by specific CTL. 76 The normal antigen peptide processing machinery, excluding the MHC antigens, was required for the peptide transfer to occur. To support the model of active peptide secretion, it was necessary to exclude CTL-mediated lysis as a source of priming peptide. This was achieved by using the L cells, which are not recognized by VSV specific CTL, as the donors. Also, TAP dependency would not be observed if peptides were transferred by viral lysis. The functioning of the secretory pathway was important as was demonstrated with the secretory block, BFA, and also required de novo protein synthesis and respiration as shown by emitine and azide. These all demonstrated that the peptide transfer was not due to a trivial release of peptide by dead cells and that it was not dependent on viral infection or the viral lytic cycle. The peptide transfer assay suggests that there is an alternative fate for lumenal peptides other than binding to MHC class I, HSPs, or transport back into the cytoplasm. Both the translocon and TAP can mediate entry into the constitutive secretory pathway. The translocon would require a signal sequence for transport into the ER whereas the TAP molecules would be involved in the transport of peptide lacking a signal sequence. Once in the ER, the two types of peptide would follow the same secretory pathway. The amount of peptides secreted would depend on the equilibrium of the peptide concentration in the ER. Peptide equilibrium in the ER would be influenced by the amount of peptides transported by TAP, the amount transported back into the cytoplasm by efflux, and the binding of peptides to HSPs and MHC class I. Overall, the peptide transfer assay described how CTL recognition of neighbouring uninfected cells presenting viral antigen was TAP mediated. 77 6. rTAPl Improves CTL Recognition of CMT.64 Cancer Cells in vivo 6.1 Introduction The previous chapter examined what effect the addition of TAP had on the CTL mediated recognition of viruses. In this chapter the role of TAP in restoring antigen presentation and immune recognition of the TAP deficient cancer cell CMT.64 was explored. Re-expression of TAP in CMT.64 cells was examined in vivo to determine whether TAP limited the emergence of tumours and malignancy related fatalities in mice. In addition, a VV containing a TAP1 gene was used to examine whether VV-TAP 1 could be used as a cancer therapy in CMT.64 burdened mice. 6.1a Cancer phenotypes Neoplastic cells arise frequently in the body due to a variety of external and internal influences and we depend on the immune system to recognize and destroy these cells before they develop into tumours. However, malignant transformations may be accompanied by phenotypic changes resulting in the ability of the cancer cell to escape the immunosurveillance mechanism. As the phenotypic changes vary with each neoplasm we are unable to develop one treatment for all cancers. Fortunately, many tumours fall into one of several larger groups of phenotypes. One such group presents with increased tumorigenicity due to a decrease in MHC class I expression (113,115,195). It was shown in a Be 13 tumour cell line that tumourigenicity of individual clones correlates inversely with the amount of H-2 class I antigens, and 78 transfection of an H-2 gene abrogated tumour growth and metastasis (196). In humans it has been estimated that 39 to 88% of tumours derived from HLA + epithelia are HLA deficient (197). Listed in table 4 are four different altered class I phenotypes proposed by Garrido et al, which summarizes the types of HLA defects found in various tumours he reviewed (197). The two most common phenotypes are total HLA loss and HLA allelic loss. For the total HLA loss phenotype the frequency of occurrence ranged from 9% of all laryngeal tumor tissues tested, to 52% of all breast tumours tested. This chapter focuses on the first phenotype in which there is a total loss of MHC class I. Table 4: Altered HLA class I phenotypes in tumour tissues Phenotype Frequency* Cause 1) total HLA loss 9-52 % defects in the antigen processing and presentation pathway 2) HLA haplotype loss rare mitotic recombination 3) HLA locus loss HLA-A - 3-19% HLA-B - 5-19% transcriptional defects 4) HLA allelic loss 15-51% HLA gene point mutations, partial deletions, chromosomal breakage * The frequency denotes what percentage the particular phenotype occurred in a range of tumours examined. The list included breast, cervical, colon, laryngeal, melanoma, pancreatic, and prostrate tumours. A decrease in cell surface expression of MHC class I can be due to a defect anywhere in the MHC class I biosynthetic pathway (197,198). There are many cellular 79 proteins that contribute to MHC class I assembly, including the proteasome, the TAP complex, MHC class I heavy chain, P2m, calnexin and tapasin, as well as other components. A deficiency in any of the antigen processing proteins may be a factor in the spread of neoplasms as the frequency of downregulation of these components is higher in metastases than in primary lesions (195). In particular, the TAP complex has been implicated in tumorigenicity of several cancers such as melanomas, cervical carcinomas, and renal cell carcinomas (195,199,200). In one study TAP downregulation was associated with a decrease in MHC class I antigen expression (66). Also, the loss or downregulation of TAP in surgically removed human lesions', was found to range from 14% in primary colorectal carcinomas to 49% in primary human papillomavirus 16 cervical carcinomas (66,195,201). These findings suggest that TAP downregulation may represent one mechanism for immune escape of malignant cells in a variety of tumours. 6.1b Antitumour effector mechanisms The immune system has evolved a very intricate recognition mechanism to eliminate diseased cells. In order for a tumour to proliferate it has to evade the cells involved in tumour recognition. The two major anti-tumour effector mechanisms are the NK cells and CD8+ CTLs (reviewed in [197]). Both cells have similar lytic mechanisms by which they deliver a lethal hit to the diseased cell through the release of perforin and granzymes (202,203). Also, both cells effector functions are controlled by MHC class I. However, in the case of CTLs the presence of MHC is essential for the 80 initiation of the effector mechanism whereas in NK cells the absence of MHC makes targets susceptible to lysis. The role of CD8 + CTLs in the recognition and destruction of class I restricted tumour associated antigens (TAA) has been well documented (114). The TAAs represent altered antigens that are not normally expressed by non-cancerous cells and they may provide the immune system with a method of detecting neoplastic cells in a syngeneic system. Since the CD8+ CTLs require the surface expression of MHC class I for the presentation of the TAAs, a MHC class I deficiency would prevent recognition by CD8+ CTLs, resulting in tumour outgrowth. As described above there is ample evidence to demonstrate that a lack of CD84- cytolysis due to MHC class I loss can be one cause of tumour growth. The immune system has also developed a way to recognize cells lacking surface MHC through the use of NK cells. The 'missing self model proposes that NK cells will kill targets lacking normal Class I expression on the cell surface. In several experimental systems it has been found that target cell susceptibility to NK lysis is inversely proportional to the level of expression of MHC class I molecules (204). NK cells are non-B or -T lymphocytes which can lyse targets without prior sensitization (205). Unlike B and T cells, NK cells do not have a single re-arranged receptor but rather a number of receptors involved in a complex signaling mechanism. These receptors interact with targets and activation of NK cells is determined by a dual recognition system of inhibitory and activating signals (206). For example, in mice NK cells are positively stimulated through the NK1.1 receptor but can be inhibited by recognition of MHC class I via the Ly-49 receptor (206). Not all of the NK receptors 81 and their ligands have been discovered, however it is believed that multiple combinations of these receptors may be coexpressed by individual NK cell clones or subsets. What influence these other receptors will have on immune recognition has yet to be determined but from what is known we can say in general that loss of MHC class I from the cell surface potentiates NK mediated lysis. Tumours with complete MHC class I loss should be resistant to CTL killing but should become susceptible to NK lysis, provided they express ligands for activating receptors. However, tumours with downregulated class I have been able to grow suggesting evasion of NK cells as well. One possible explanation for this conflict was put forward by Garrido et al, and is related to tumour evolution (197). In this model tumours would be selected for a minimum loss of MHC to permit CTL evasion without inducing NK susceptibility. As MHC downregulation is not exactly correlated with NK susceptibility the CD8+ mediated recognition of tumours needs to be explored further. In this thesis the role of TAP in CTL mediated killing of tumours was explored using the CMT.64 cell line, which has downregulated MHC class I. 6.1c The CMT.64 cell line The CMT.64 cell line is a small cell lung carcinoma (SCLC) which arose spontaneously in a C57B1/6 mouse (119). The CMT.64 cell line is lacking in both MHC class I expression and endogenous antigen presentation. IFN-y treatment corrects these deficiencies. However, the underlying defect remains unknown (207,208). CMT.64 shows decreased expression of MHC class I heavy chain, p2m, proteasome components LMP2 and LMP 7, and TAP1 and 2 (89). Restoration of p2m to levels 82 seen in IFN-y induced CMT.64 cells does not improve CTL recognition of viral peptides, suggesting lack of p2m was not the primary deficiency (89,120). Several lines of evidence demonstrated that the MHC class I protein itself was not the defect in CMT.64. First, immunoprecipitation, and pulse chase experiments showed that H-2Kb and H-2Db were synthesized by CMT.64 but failed to obtain the higher molecular weight form characteristic of transport out of the ER (120). In uninduced CMT.64 MHC class I is barely detected on the cell surface. However, the MHC class I was functional as IFN-y treatment restored class I cell surface expression and viral presentation. Finally, in a CTL assay addition of exogenous H-2Kb specific VSV or influenza peptide restored viral presentation in uninduced CMT.64 demonstrating that some MHC class I is transported to the cell surface and can present peptide (120). The supply of peptides by TAP to the MHC class I was then investigated. It was shown that TAP1 and 2 levels were undetectable in CMT.64 by northern blot analysis unless the cells were treated with IFN-y (89). Although the CMT.64 cell is very poor at antigen presentation, reconstitution with rTAPl and 2 increases cell surface MHC class I to levels that are sufficient for the restoration of viral antigen presentation in vitro (89,120). The increase in MHC class I levels were never as high as seen in IFN-y treated cells due to limiting amounts of other assembly proteins. However, restoring all the components to wild type levels is not required for recognition of antigen by T lymphocytes (120). These data demonstrated that the blockage of MHC class I assembly was at the level of transport of peptide from the cytoplasm to the ER. 83 It is difficult to treat cancers since many of them have multiple cellular defects. However, if restoring one component will allow the hosts immune system to recognize and destroy the cancer, then the cancer treatment would be less complicated. Many of the tumors lacking surface MHC class I have TAP losses or dysfunctions (195). If CMT.64 transfected with TAP is sufficient for restoring antigen presentation in vitro then perhaps the immune system could now recognize the cell, destroy it and thus prevent metastasis. To examine this possibility, the TAP deficient CMT.64 as well as the TAP transfected cell lines derived from CMT.64, were tested to see if TAP could improve the immune response against the cancer cell and thus improve survival of the animal bearing this tumour. 6.2 Results 6.2a The TAP complex improves immune recognition in vivo When injected intraperitoneally (ip.) into syngeneic mice CMT.64 spreads throughout the mouse. A tumour load of 5 X 105 cells will begin to kill the mice in 3 weeks with a TD 5 0 (time at which 50 % are dead) of approximately 28 days. The CMT.64 cell lines transfected with rTAP are derived from the transfection of CMT.64 with the mammalian plasmid pHp" Apr-1 neo containing the rTAP genes (119,120). To confirm that components in the plasmid did not alter the survival of syngeneic mice injected with the SCLC, an in vivo comparison was made between the vector only control, CMT.neo, and CMT.64 (figure 16). Although the TD 5 0 for CMT.neo was 32 days versus 28 days for CMT.64, the difference between these two cell lines is 84 100 -» 90 80 70 60 -I S 50 40 30 J 20 10 20 — i — 40 - A — CMT.64 - B — CMT.neo -m 60 — i — 80 100 Time after injection (days) Figure 16: Comparison of the survival of mice injected with CMT.64 or the vector only control CMT.neo. Two groups of 14 mice were injected ip. with either 5X105 CMT.64 or CMT.neo in PBS. Time of death of the mice was recorded. 85 Table 5: Surface MHC class I expression by CMT.64 and CMT.neo Cell Line Db K b CMT.64 31 3 CMT.neo 52 28 CMT.64 + IFN-y 1100 590 FACS analysis of Kb and Db expression were performed on the CMT.64, CMT.neo, and CMT.64 treated with IFN-y for 48 hours. The monoclonal antibodies Y-3 (Kb) and 28.14.8S (Db) and secondary antibodies were incubated at 4°C. The results are the mean linear fluorescence following subtraction of negative controls. Results are from Gregor Reid. statistically insignificant (p=0.114) demonstrating that the plasmid did not alter immune recognition of CMT.64. Also, the presence of the pHpApr-1 neo plasmid did not significantly increase the surface MHC class I levels as seen in a FACS analysis (table 5). The MHC class I levels on CMT.neo is slightly higher than in CMT.64 but 21 fold lower than CMT.64 treated with IFN-y. The TAP complex functions as a heterodimer and previous research has shown that despite CMT.64 being deficient in several of the antigen processing and presentation components, reconstitution of CMT.64 with both rTAPl and 2 (CMT12.12) can restore antigen presentation for recognition by CTL (120). We investigated whether CMT.64 transfected with both rTAPl and 2 (CMT12.12) could present antigen at the cell surface, therefore allowing recognition by the host immune 86 system. The ability of rTAP12 to improve the recognition of CMT.64 was determined by comparing the survival of mice injected with the various cancer cells. An improvement in survival would suggest an increased recognition and destruction of the cancer cell. As was demonstrated in figure 17 the mice in the CMT12.12 group did not survive longer than the CMT.64 group (p=0.441, TD50=37 days). These results did not demonstrate whether the lack of recognition was due to the inability to present a 'foreign' peptide, or that the MHC levels were not high enough to elicit a sufficient immune response. The current theory states that both TAP1 and TAP2 are required to form a functional TAP complex. However, there is research demonstrating that in some cancer cell lines only one of the TAP molecules is required for viral antigen presentation in vitro. Once such example is the murine lymphoma RMA-S which does not contain a functional TAP2 molecule yet it can still present certain viral peptides (209,137). Similar results are seen in CMT.64 cells transfected with either rTAPl or 2 which can present viral antigens to CTL (89,120). To confirm this data, a VSV specific CTL assay measuring viral presentation was performed with CMT.64 cells transfected with individual rTAPs (figure 18). CMT.64 was unable to present the VSV N peptide whereas both the rTAPl (CMT1.4) and rTAP2 (CMT2.10) transfectants presented peptide to VSV specific CTL. Moreover, there was a 9 fold difference in lysis between CMT1.4 and CMT2.10, as 17-18% lysis for CMT2.10 was achieved at an E:T ratio of 90 whereas the same amount of lysis for CMT1.4 was achieved at an E:T ratio of 10. 87 Figure 17: rTAP12 transfected CMT.64 cells do not survive longer than the TAP deficient CMT.64 cells. 5X105 cells in PBS of the TAP deficient cell line, CMT.64, or CMT.64 transfected with rTAPland 2, (CMT12.12 ) were injected ip into 15 C57B1/6 syngeneic mice. The time of death after injection was then recorded. 88 Figure 18: Viral presentation by CMT.64 transfected with single TAPs A VSV specific CTL analysis was performed on CMT.64 cells transfected with either rTAPl (CMT1.4) or rTAP2 (CMT2.10) to determine whether the presence of single TAPs restored viral antigen presentation. The CMT targets were infected (MOI = 10) with VV-NP for 24 hours.The presentation of VSV N peptide was determined through target cell lysis by VSV specific CTL in a 4 hour 51Cr release assay. 89 Since in vitro viral presentation was restored with the expression of single TAP molecules into CMT.64, we investigated whether CMT1.4 and CMT2.10 were recognized better by CTL in vivo. Mice were injected ip with either CMT1.4 or CMT2.10 and survival rates were compared to those of CMT.64 recipients (Figure 19). At 100 days post injection, 60% of the CMT1.4 (p<0.001) group were still alive, demonstrating that the presence of rTAPl was sufficient for improving immune recognition. In contrast, the CMT2.10 group containing rTAP2 (p=0.210) did not demonstrate any difference in survival compared to CMT.64. Autopsies were performed on the different groups of mice and the tumours ranged from a few larger tumours (1-2 cm.) to many smaller tumours (0.2-0.9 cm.). The number of mice presenting with many small tumours was greater in the CMT.64, CMT2.10, and CMT12.12 groups, whereas the CMT1.4 group had a few larger tumors or no tumors at all. Since the greatest difference was seen in the CMT1.4 group a representation of tumours found in mice autopsied is given in table 6 in order to compare the difference in tumours seen in CMT64, CMT.neo, and CMT1.4. The mice that died in the first month had an average of 15 small (0.2-0.5 cm.), 6 medium (0.6-0.9 cm.), 0.05 large (1-2 cm.) tumours per mouse for CMT.64 and 27 small, 3 medium tumours for CMT.neo. In the second month, there were less tumours overall that were in general larger (12 small, 2 medium, 1 large for CMT.64 and 4 small, 9 medium and 0.3 large for CMT.neo). In contrast, 63% of the CMT1.4 injected mice had no tumours. Also, the CMT1.4 mice that died in the second month had less tumours per mouse (1 small, 3 medium, 0.5 large) in comparison to the CMT.64 and 90 120 0 -I , , 1 , , , , 0 20 40 60 80 100 120 Time after injection (days) Figure 19: Improved survival of mice receiving TAP1 transfected CMT.64. 5X105 cells in PBS of CMT.64, or 5X105 CMT1.4 expressing rTAPl alone, or 5X105 CMT2.10 expressing rTAP2 alone were injected ip. into 15 C57B1/6 syngeneic mice. The time of death after injection was recorded. 91 Table 6: Comparison of the tumor number and size in mice injected with CMT.64, CMT.neo, or CMT1.4. 1st Month 2nd Month > 3 Months Group #of small med. large #of small med. large #of small med. large mice mice mice CMT1.4 - - - - 1 5 1 1 - 5 -1 10 1 1 1 1 10 a 1 1 total - - - - 4 5 11 2 12 - 6 -CMT.64 7 >30 2 >30 1 3 - -2 >20 2 >20 l b 2 >5 1 >15 5 >20 1 3 1 15 1 >25 1 1 5 1 1 >20 1 1 4 1 3 1 2 1 1 total 18 >265 >115 1 12 >143 >20 12 2 3 - -CMT.neo 2 >50 1 >20 - - - -2 >30 4 >10 >10 1 >25 1 >10 >5 1 >30 >20 1 >30 2 >20 2 2 >20 1 >15 1 1 >15 1 3 3 >10 1 3 1 1 5 1 2 2 1 1 total 10 >270 >28 - 18 >70 >165 5 - - - -a= no tumours found, mice did not die b=no tumours found The data is a compilation of results from autopsies done on groups of mice from several experiments. The mice are placed in the month column in which they died. The autopsy was a visual count of the number of tumours in the peritoneal cavity. The tumour number for each mouse was entered under the columns labeled as small (0.2-0.5 cm), medium (0.6-0.9 cm), or large (1-2 cm), and a running total was kept for each group. 92 A Figure 20: Comparison of CMT.64 rTAP transfectants with CMT.neo. A) 5X105 CMT.neo, or CMT1.4, or CMT1.10, or CMT2.1 in PBS were injected ip. into 14 C57B1/6 mice. B) 5X105 CMT.neo, or CMT12.12, or CMT12.21 in PBS were injected ip. into 14 C57B1/6 mice. Time of death was recorded. 9 3 CMT.neo injected mice. The autopsy results demonstrate that the inclusion of TAP1 resulted in fewer tumours which were in general larger. Table 6 also illustrates over several experiments the enhanced survival of the CMT1.4 mice over the CMT.64 mice. In the first 30 days after injection of the tumour cell 56% of the CMT.64 mice and 36% of the CMT.neo injected mice died in comparison to zero deaths in the CMT1.4 group. None of the CMT.neo group and only 6% of the CMT.64 mice survived longer than 2 months after injection. In contrast, 75% of the CMT1.4 mice were still alive in the third month. This demonstrates that re-expression of TAP in CMT.64 improves mouse survival. The differences in survival between CMT1.4, CMT2.10, and CMT12.12 could be due to clonal variation. To address this possibility, the experiment was repeated along with a second set of CMT-TAP transfectants (figure 20). The CMT.64 cell line containing rTAPl (CMT1.4: p=0.012, TD50=56, CMT1.10: p=0.014, TD50=65) again demonstrated a significant increase in survival times over CMT.neo (TD50=32). Once again the rTAP2 clone, CMT2.1 (p=0.095, TD50=30), and the double transfectants, CMT12.12 (p=0.421, TD50=30) and CMT12.21 (p=0.049, TD50=30), showed no increase in survival over the mice receiving CMT.neo (TD50=32). 6.2b The nature of tumour recognition A cell mediated response against tumours is influenced by the expression levels of MHC class I as well as the expression of tumour associated antigens (196,210). Neoplasms that are unable to display tumor antigens are not be killed. It is important that surface MHC levels are sufficiently high for the display of the antigens. In table 7, 94 FACS analysis shows that although the TAP transfectants have higher levels of surface MHC class I than CMT.64, they are all significantly lower than the IFN-y treated CMT.64 (120). The reason the MHC levels on the transfectants do not approach the IFN-y treated levels might be because of other deficiencies in antigen processing components such as the MHC class I heavy chain or P2m, which could limit the amount of MHC class I available to bind peptide (89). The single TAP1 transfectants which have the highest TD 5 0 also have the highest levels of surface MHC class I. The CMT12.12 cells, and single TAP2 transfectants have the lowest TD 5 0 as well as the lowest levels of MHC. This suggests that the amount of surface MHC is an important factor in the survival of the mice. However, this minimum MHC level does not appear to hold true for CMT12.21. Although it behaves similarly to CMT12.12 (TD50=30 for both) in the in vivo cancer studies it has MHC levels closer to CMT1.10. When comparing the two TAP1 transfectants, CMT1.10 has 50% less MHC than CMT1.4, yet CMT1.10 has a similar survival time. This may indicate for this cancer model that along with a minimum level of surface MHC, the peptides which are presented are also important for tumour recognition. The TAA, if it exists, for CMT.64 is unknown. Therefore we cannot determine if the TAP molecules were able to restore presentation of the antigen. Nevertheless, we investigated whether CMT 1.4 was presenting an antigen that CMT.64 was not. A CTL analysis was performed using splenocytes from mice which had been injected with CMT.neo or CMT1.4 (figure 21). Splenocytes from mice injected with CMT.neo recognize CMT.neo and CMT1.4 targets equivalently. On the other hand, splenocytes 95 Table 7: Comparison of MHC class I surface expression with TD 5 0 in the CMT-TAP transfectants Cell Line Db K b TD 5 0 CMT.64 18 0 28 CMT.64 + IFN-y 1100 590 — CMT1.4 164 64 56 CMT1.10 68 36 65 CMT2.1 10 3 30 CMT2.10 39 48 35 CMT12.12 49 9 30 CMT12.21 103 22 30 The TD 5 0 data are compiled from the tumour experiments presented so far. FACS analysis of Kb and Db expression were performed on the CMT.64 and the CMT.64 TAP transfectants. The monoclonal antibodies Y-3 (Kb) and 28.14.8S (Db) and secondary antibodies were incubated at 4°C. The results are the mean linear fluorescence following subtraction of negative controls. FACS results are reproduced from reference 120. 96 A 25 20 .2 15 to >% i <• 5 0 fcfct 100 50 25 12 6 Effector:Target Ratio I CMT.neo ICMT1.4 B en in 25 20 15 10 5 0 100 . • 50 25 12 6 Effector:Target Ratio _ • 3 I CMT.neo ICMT1.4 Figure 21: Specificity of splenocytes from mice injected with CMT.neo or CMT1.4 The specificity of splenocytes from a mouse injected with A) CMT.neo or B) CMT1.4 was determined in a CTL assay against the 51Cr labeled targets CMT.neo, and CMT1.4. Upon removal the splenocytes were cultured with stimulator cells at a 3:1 ratio. The stimulator cells were prepared by incubating CMT1.4 or CMT.neo cells with 30 ug/ml mitomycin C under hypoxic conditions. After a 2 hour incubation the cells were y-irradiated (10,000 Rads) and washed three times before addition to the splenocyte culture. CMT.neo splenocytes received CMT.neo stimulators, whereas CMT1.4 splenocytes received CMT1.4 stimulators. 6 days after in vitro stimulation the splenocytes were tested in a standard 4 hour 51Cr release assay. 9 7 from mice injected with CMT1.4 recognized CMT1.4 better than CMT.64. This data suggests that CMT1.4 presented an antigen from CMT.64 which is being recognized by the host immune system but is not being presented by CMT.64. Since we are using TAP to increase the CTL mediated cytolysis, it was important to determine whether the survival of the CMT1.4 mice in vivo was due to specific CTL lysis. The fact that CMT1.4 has surface MHC levels greater than CMT.64 suggested that an increase in antigen presentation to T cells was possibly a factor in the survival of the CMT1.4 mice. To examine the influence of cytotoxic T cells on survival, nude mice which are devoid of a T lymphocyte system, were used (211). If the destruction of the CMT1.4 cells in mice was mediated by T cells then the CMT1.4 and CMT.64 mice would have the same survival rates. As seen in figure 22, mice which received CMT1.4 (p=0.037, TD50=22 days) died just before the CMT.64 treated mice (TD50=26 days) suggesting that specific CTL lysis was responsible for the recognition and destruction of the CMT1.4 cells in normal mice. Although the CMT1.4 mice demonstrated an improved survival over the CMT.64 mice, the improvement was marginal and could be attributed to the low number of mice used in this experiment. The fact that the survival of the CMT1.4 mice dropped from a TD50=56 days in normal mice to 22 days in nude mice demonstrates the influence of the T cells on survival and argues against NK cells, which are hyperactive in nudes. 98 Figure 22: TAP does not improve recognition of CMT.64 in nude mice 5 X 105 cells of either CMT1.4 (5 mice) or CMT.64 (4 mice) were injected ip into nude mice (H-2b). The time of death was recorded. 99 6.2c Utilizing TAPs in cancer therapy Some neoplasms grow because they do not trigger an immune response, yet an effective immune response can be elicited by immunizing against tumour antigens (212,213). In melanoma cancer therapy Marchand et al were able to isolate a melanoma antigen and immunize the patient (214). The results presented in this thesis so far, have demonstrated that TAP1 re-expression in CMT.64 results in enhanced survival of tumour bearing animals (figures 19, 20). This suggests that it may be possible that 'foreign' antigens are being displayed which could prime the immune system to recognize low antigen levels on the CMT.64 cell. Immunization with live cancer cells can not be done since it would unnecessarily introduce a cancer into the individual. However, if it is possible to prime the immune system to see antigens on CMT.64 then perhaps we could immunize with killed whole cell CMT TAP transfectants (figure 23). To test this hypothesis 107 cells of CMT.64 or the TAP transfectants were killed by treatment with mitomycin C, and y-irradiation before ip. injection into the mouse (215,216). One month later the mice were challenged with 5xl05 CMT.64 cells. In comparison to the CMT.64 primed mice (TD50=40 days) only the single rTAPl transfectant 1.10 (p=0.045, TD50=47 days), was able to improve survival against CMT.64. However CMT 1.4 (p=0.168, TD50=40), CMT.neo (p=0.789, TD50=37 days), CMT2.1 (p=0.448, TD50=37 days), CMT2.10 (p=0.071, TD50=41 days), CMT12.12 (p=0.594, TD50=38 days) and CMT12.21 (p=0.320, TD50=36 days) were unable to provide any protection. Only one of the TAP1 transfectants was able to provide some significant protection which suggests that TAP1 was presenting an antigen but it was weakly immunogenic. 100 CMT.64 - - - CMT.neo 20 25 30 35 40 45 50 55 Time after challenge (days) 101 c (0 > E 3 (0 CMT.64 CMT1.4 CMT1.10 35 40 45 Time after challenge (days) 60 D CMT.64 CMT2.1 - - - CMT2.10 Time after challenge (days) Figure 23: Immunization with the CMT-TAP transfectants 107 cells of A) CMT.64, or CMT.neo, B) CMT12.12, or CMT12.21, or C) CMT1.4, or CMT1.10, or D) CMT,2.1, or CMT2.10, were treated with Mitomycin C (30ug/ml) for 2 hours and y-irradiated (10,000 Rads) before injection ip into 10 C57B1/6 mice. One month later the mice were challenged ip with 5 X 105 CMT.64 cells in PBS. Time of death was recorded. 102 In cancer therapy the opportunity to immunize against a cancer is rare since it is difficult to predict which cancer a person will get, and it is often difficult to immunize against the cancer once it has established itself. More importantly, while benign tumours can be easily excised, for a metastatic cancer it is impossible to remove all of the cancerous cells. MHC class I loss has been found to be indicative of a more aggressive phenotype and more rapid tumor growth (196,217). For these reasons cancer therapies need to be developed for when there is an existing tumour burden that 'can not be surgically removed. To see if TAP could have a role as a possible therapy the VV-rTAPl construct was used to see if it could improve survival in a CMT.neo tumour burdened mouse (figure 24). The first experiment (figure 24a) compares mice receiving a tumor load of 5xl05 CMT.neo cells ip to mice receiving the same number of cells pre-infected with either VV-pJS5, or VV-rTAPl (MOI=0.1). Pre-infection eliminates variability in an infection, and allows the direct testing of the effect of the TAP molecule would have over any effects received from the VV infection itself. The MOI used will not infect all of the cancer cells but will infect enough cells to allow a determination of whether or not the VV-rTAPl has an effect over the vector only control VV-pJS5. The data demonstrates that the VV-pJS5 group of mice survived longer than the mice receiving only CMT.neo (p<0.01). The VV-pJS5 group of mice denotes the basal level at which the VV antigens are recognized by the immune system. Mice that received VV-rTAPl had a higher level of survival than VV-pJS5 infected mice (p=0.04 by day 41), implying that perhaps the TAP was providing foreign VV antigens to the MHC class I which are being recognized by the mouse. The group of mice that received VV-rTAPl showed improved survival over VV-pJS5 levels by day 103 A 120 -J 100 • _ 80 1 60 -I « 40 20 -I 0 1 10 20 30 40 50 60 Time after injection (days) — i — 70 •CMT.neo W-pJS5 W-rTAP1 80 90 B 120 100 80 H 60 <" 40 20 0 —CMT.neo — W-pJS5 - W-rTAP1 —r-20 V -— I — 40 — i — 60 — i — 80 100 Time after injection (days) Figure 24: W-rTAPl therapy of CMT.neo burdened mice Three groups of 10 C57B1/6 mice were injected ip. with 5X105 CMT.neo in PBS. Two of the three groups received either VV-pJS5, or VV-rTAPl treatment. In A) the rVV treated CMT.neo cells were pre-infected with rVV (MOI=0.1) before injecting the cells into mice. B) In the two rVV treated groups, the CMT.neo cells were injected into the mice then treated ip. at 24 hours, and at 2 weeks with 106 pfu rVV in PBS containing 2% C57B1/6 mouse serum. 104 41 (p=0.036). The increase in survival of the VV-rTAPl group may be due to increased antigen presentation as a consequence of the presence of TAP. The viral therapy of a tumor burdened individual would not involve the use of preinfected cells. Instead it would entail an in vivo infection after the tumour load had been established. To examine this scenario, 5xl05 CMT.neo cells were injected into 3 groups of mice. While one group received no treatment the other two groups were injected with either VV-rTAPl or VV-pJS5 24 hours later and again at 2 weeks (figure 24b). As expected, the vector only control, VV-pJS5, (p=0.18) did not demonstrate any improvement over the group of mice injected with CMT.neo. However, the mice receiving VV-rTAPl survived longer than the CMT.64 group (p=0.01), and the VV-pJS5 treated mice (p=0.04). In order to determine whether the mice that received VV-rTAPl showed improved survival due to presentation of a CMT.neo antigen or to increased VV antigen presentation, a CTL analysis was performed using the splenocytes from mice injected with CMT.neo + VV-rTAPl or CMT.neo + VV-pJS5. The splenocytes from the mice injected with CMT.neo + VV-pJS5 lysed the CMT1.4 target that was infected with VV-pJS5 but not the other targets (Figure 25). This demonstrates that the mice could mount an immune response to VV during the treatment. The level of the response of splenocytes from the mice treated with VV-rTAPl was tested to see if they recognized the CMT1.4 + VV-pJS5 targets better. If the splenocytes kill the VV-pJS5 infected target better than CMT1.4 then the improved survival of the VV-rTAPl treated mice would have been due to an increased presentation of VV antigens when 105 1 100 50 25 Effector:Target Ratio 12 g CMT.neo gCMT.neo+W-pJS5 raCMT1.4 • CMT1.4+W-pJS5 Figure 25: Specificity of splenocytes from CMT.neo + VV-pJS5 mice. The specificity of splenocytes from a mouse injected with CMT.neo and VV-pJS5 was determined in a CTL assay against the 5 lCr labeled targets CMT.neo, CMT1.4, and VV-pJS5 infected CMT 1.4 or CMT.neo (MOI = 10, 3.5 hours). The splenocytes were cultured with stimulator cells at a 3:1 ratio. The stimulator cells were prepared by infecting CMT.neo cells with VV-pJS5 (MOI=2) overnight before adding 30 ug/ml mitomycin C under hypoxic conditions. After a 2 hour incubation the cells were y-irradiated (10,000 Rads) and washed three times before addition to the splenocyte culture. After a six day incubation the splenocytes were tested in a standard 4 hour 5 lCr release assay. 106 B CMT.neo HCMT1.4 • CMT1.4 + W-pJS5 100 50 25 12 Effector.Target Ratio Figure 26: Specificity of splenocytes from CMT.neo + W-rTAPl mice. The specificity of splenocytes from two mice injected with CMT.neo and VV-rTAPl was determined in a CTL assay against the 5 lCr labeled targets CMT.neo, CMT1.4, and VV-pJS5 infected CMT1.4 ( MOI=10, 3.5 hours). The splenocytes were a secondary mass culture which were cultured with stimulator cells, plus y-irradiated (5,000 Rads) naive C57B1/6 splenocytes, at a 5:1:15 ratio. The stimulator cells were prepared by infecting CMT.neo cells with VV-rTAPl for 3 hours before adding 30 p-g/ml mitomycin C under hypoxic conditions. After a 2 hour incubation the cells were y-irradiated (10,000 Rads) and washed three times before addition to the splenocyte culture. After a six day incubation the splenocytes were tested in a standard 4 hour 5 lCr release assay. 107 TAP was present. However, figure 26 shows that both CMT1.4, and VV-pJS5 infected CMT1.4 were killed equally well. Therefore the improved survival may be due to the presentation of CMT antigens and not VV antigens. 6.3 Discussion The TAP complex is a very important component in the antigen processing pathway and without it surface MHC class I is downregulated (64-68). The immune system's ability to recognize a cancerous cell is profoundly reduced if the cell lacks surface MHC class I (66,72-74,195). For cancer cells, this may lead to unchecked growth of tumour cells. There are many cancers which have downregulated MHC class I, of which CMT.64 is one (195,197). The lack of MHC class I cell surface expression in CMT.64 is due to a deficient peptide supply by TAP (89,120). TAP deficiencies have been correlated with increased tumourigenicity in a variety of cancers (195,218,219). In order to study whether TAP downregulation plays a role in tumourigenicity in vivo studies were undertaken to see if reconstituting CMT.64 cells with rTAP would restore immune recognition and improve survival. rTAPl alone significantly improved survival whereas rTAP2 alone, and rTAPl and 2 together did not. As the TAP complex functions as a heterodimer it was surprising that the addition of rTAPl improved mouse survival. Since normal levels of MHC class I can be restored with IFN-y treatment in vitro, possibly low levels of IFN-y are present in the in vivo experiment (120). However, it is unlikely that IFN-y has much of an effect since CMT.64 escapes the immunosurveillance mechanism and in in vitro systems 108 where IFN-y is not present, single TAPs restore antigen presentation (120). However, if there were low levels of IFN-y there could be some endogenous amounts of murine TAP1 and 2 produced which would allow peptide transport by the rat TAP 1: mouse TAP2 complex. It would be interesting to see if the same results could be achieved with transfection of TAP1 into a non-inducible TAP deficient cancer cell. Overall, this data demonstrated that TAP1 alone was sufficient for improving survival. It is suprising that the CMT.64 cell transfected with TAP 12 did not improve mouse survival as well as the single TAP1 transfectant. There are several possible explanations for this result. First, there could be differential peptide transport in the single TAP versus the double TAP transfectants. The transfectant data of figures 19 and 20 suggest that the rTAPl protein is very important for the transport of an antigenic peptide for in vivo destruction of the CMT cell. However, the levels of rTAPl in the single transfectant may be higher than the levels found with TAP12. If TAP functions as a heterodimer then you would expect improved survival in CMT12.12 but not in CMT1.4. However, evidence exists for the possible formation of TAP1 homodimers (90,137,209). In this case there would be more TAP1 complexes in the single transfectants as some of the TAP1 in the double transfectants would be complexed with TAP2. The existence of homodimers is controversial. Cell lines, like T2, can not restore viral presentation after transfection of a single TAP subunit whereas RMA-S which does not have a functional TAP2 can present influenza and VSV peptides (64,66,73,120). The phenotype for T2 could be due to the fact that it 109 contains large deletions in its MHC region which knock out several proteins involved in the antigen processing and presentation pathway (220). A second possible explanation relates to the different peptide pools which could be transported by different rat and mouse TAP combinations in the various transfectants. On the one hand it is presumed that the TAP transporters have a broad peptide specificity yet it has also been shown that a single mutation in TAP2 can severely alter selectivity (221). To date the fine specificity of peptide transporters has not been fully discovered, and only a small population of peptides have been tested. So, if mouse TAP was available in CMT transfectants due to local endogenous IFN-y being present then it could be that the rat TAPI:mouse TAP2 combination transports different peptides than the rat TAP 12 complex. In the double transfectants the majority of rat TAP1 could be combined with the rat TAP2 since they would presumably be produced at higher levels than the endogenous mouse TAP complex. The peptides transported by the dominant rat TAP complex may out compete peptides transported by other minor TAP combinations present in the cell, and the peptides transported by the rTAPl:mTAP2 complex would not have a significant effect. In the single TAP1 transfectant the peptides transported by the rTAPl :mTAP2 would comprise the majority of the peptides which the MHC class I would see. One final explanation for the TAP1 transfectant being more effective than the TAP12 transfectant could be due to some heretofore undiscovered factor/protein which interacts with TAP1. The TAP complex affects the specific immune response in two ways. First, it enforces some selection on the peptides that can bind to MHC class I, and secondly, it maintains a continuous supply of peptides for the MHC biosynthetic pathway. In these 110 experiments we saw that both of these factors may be important for the enhanced survival of mice injected with CMT1.4. The surface MHC class I levels were sufficient for in vivo antigen presentation as seen in the TAP1 transfectants, but in one of the double TAP transfectant clones (CMT 12.21) which had similar levels of surface MHC to a TAP1 clone (CMT1.10), the mice did not survive longer. This suggests that both criteria for a functioning TAP complex must be met for the improved recognition of the CMT.64 cell. There has to be a pool of peptides in the ER lumen so that there can be an increase of MHC on the cell surface and it is important which immunogenic peptide is being supplied by the TAP complex. A particular peptide may not need to be in high concentrations if it is more immunogenic. In this system, any influence imparted by MHC polymorphism would be negligible since it remains the same in all experiments. If these peptides were not part of the normal repertoire presented by the MHC class I then they may be seen as foreign and therefore the cell killed. So we could suggest for CMT.64 that the type of peptides transported is as important as the amount of peptides provided by TAP. There are a wide variety of cancers and many of them are deficient in TAP complexes (66,195,199,199,201). Due to the many different types of cancers it is impossible to immunize against them all. It is more common to have a patient present with a tumor and then use some form of therapy to eradicate the cancer. This has been seen with some success, for example, in melanomas where the MART-1 TAA has been used for immunization (222). The research done here suggests that the TAP2 and TAP12 transfectants would be unlikely candidates for immunization. Since CMT1.4 111 and CMT1.10 injected into mice improved survival of the mice over CMT.64, it was expected that the TAP1 transfectants would provide some protection in the immunization experiment. However, the protection with CMT1.4 was not significant and only marginally better for CMT1.10. The fact that CMT1.10 did provide some protection suggests that there may be some immunogenic peptide being presented. The poor immunogenicity of CMT 1.4 and CMT 1.10 in the immunization experiments is in contrast to the ability of TAP1 to sensitize CMT.64 to CTL mediated lysis in the tumour experiments (figures 19 and 20). However, this is not an uncommon phenomena. Kundig et al demonstrated that the murine thymoma, EL-4, transfected with the VSV N protein (EL-4NP) was poorly immunogenic despite its ability to elicit a strong CTL response (223). It was determined that the EL-4N P cells were unable to induce sufficient NP-specific T cell help because the costimulatory or 'second' signal required for activation of T cells was absent. CMT1.4 and CMT1.10 may be presenting immunogenic antigen as suggested by the tumour and CTL data however they may be unable to activate a T cell sufficiently. A variety of methods have been created to elicit an anti-tumour response with non-immunogenic tumours such as transfecting tumours with IL-2, or the costimulatory ligand B-7 (224-226). So, the potential exists for using TAP to immunize against certain cancers but it may require additional factors, such as B7. The potential of using TAP in a viral vector as a therapy for tumour burdened individuals provides a possible method of treating metastasized neoplasms. This would be particularly true for the cancers which have a downregulated MHC class I phenotype. Utilizing VV allows the TAP to be produced in any cell of the body and 112 that using TAP in this manner provides a possible treatment for improving survival. The use of VV is not essential, as many methods of getting specific proteins to be expressed in different cancer cells are being developed. Ultimately, targeting TAP only into cancer cells would be preferred so that the risk of autoimmunity would be reduced. 113 7. General Conclusions It is clear from this and other studies that the TAP complex is an important component of the antigen processing and presentation pathway. Cells deficient in TAP have decreased levels of MHC class I on the cell surface which has serious implications concerning disease onset and progression in the areas of cancer, viral infection, and other endogenous pathogenic infections. If the body is unable to recognize a diseased cell it has no way of eliminating it and a diseased state progresses. Based on this information it was interesting to examine the effect TAP had on CTL mediated killing. In this thesis, several situations were examined where CTL killing was involved. First of all, it was demonstrated that TAP could act as an adjuvant and enhance a CD8 + immune response against a peptide. It was also determined that viral peptides could be secreted in a TAP dependent manner from a virally infected cell and sensitize neighbouring cells to specific CTL lysis. Finally, the restoration of TAP in cancerous cells deficient in TAP resulted in improved tumour recognition and greater survival of the mice. 7.1 Viral antigen presentation We have seen that including TAP as an adjuvant in a subunit vaccine which contains a cytotoxic epitope elicited a greater T cell response in immunized mice. It was so effective that drastically reduced levels of antigen were still effective in evoking a strong immune response. It has been suggested that the minimum number of MHC:peptide complexes on the cell surface required to initiate an immune response is between 100 to 400. Kageyema et al. demonstrated that as few as 10 complexes were 114 sufficient (227-231). Also, in CTL assays there is a corresponding increase in target lysis as the concentration of specific peptides increases (89,183). Thus, the more antigenic peptides on the cell surface the more likely the TcR-MHC interaction will result in the activation of a T cell. It is believed that one of the limiting factors in the amount of MHC class I on the cell surface can be the supply of peptides in the ER (94,95). As the TAP complex is responsible for the supply of peptides we investigated whether increasing the number of TAP molecules in the cells will increase the amount of peptide displayed on the cell surface. In vitro CTL studies demonstrated no increase in lysis when WT cells were given more TAP, however this result cannot distinguish whether peptide supply was limiting to TAP or to MHC class I. It is not known whether the supply of peptides for MHC class I is limiting due to a low cytoplasmic supply of peptides for TAP to transport or as a result of the optimal TAP transport efficiency. Figure 27 depicts the possible outcomes of increased TAP expression. There are two situations in which increasing TAP above WT levels would not make a difference. If the cytoplasmic peptide supply for TAP is limiting then increasing the number of TAP complexes in a WT cell would not increase lysis. Also, no increase in lysis would be seen if the supply of peptides was saturating for both TAP and MHC class I. However, viral infections produce large amounts of viral protein, so unless proteases were deficient there should be large amounts of peptide available for TAP suggesting peptides were saturating for both TAP and MHC class I. The RMA augmentation assay was unable to discern why there was an increase in the immune response against the VSV N peptide but perhaps the answer could be found in 115 normal TAP levels ^ ^ X ^ same level Cytoplasmic peptides limiting ^ of lysis extra TAP levels B peptides saturating and TAP operating at maximum peptides limiting for MHC peptides saturating for MHC normal TAP levels extra TAP levels normal TAP levels extra TAP levels higher lysis with extra TAP levels same level of lysis C peptides saturating . peptides . peptide . more cells and TAP operating saturating secretion displaying at maximum for MHC & transfer antigen Figure 27: Possible model for TAP adjuvant effect. In the RMA augmentation assay the following possibilities exist: A) If peptide supply to TAP was limiting then increasing TAP would not increase antigen presentation. B) If peptide supply is saturating then additional TAP would increase lysis only if peptide supply for MHC class I was limiting in the ER. In the peptide transfer assay C) the second model would hold as supply is saturating for TAP and MHC so peptides are secreted, which results in neighbouring cells also displaying the antigen. 116 the peptide transfer assay. The peptide transfer assays have demonstrated that there can be an alternative fate for the antigenic peptides. It also suggests a role for this alternate fate in contributing to the magnitude of a cytotoxic immune response against a peptide. At first it was believed that peptides that did not bind to the MHC class I in the ER lumen, because of incorrect peptide binding motif or if they are in excess, were rapidly degraded (109,110). However, it has been demonstrated that these peptides may be bound to heat shock proteins, or be transported back into the cytoplasm by efflux (93,111,112). The peptide transfer assay showed that ER peptides not bound to MHC class I could also be secreted out of the cell through the constitutive secretory pathway in a TAP dependent manner. This assay did not demonstrate the form in which they were secreted. For instance, they could be attached to a carrier protein. But the assay did show that antigenic peptides that are secreted from the cell could sensitize neighbouring cells to CTL lysis. The peptide transfer mechanism fits into the model suggested in Figure 27. Viral proteins would presumably be saturating TAP complexes. If MHC class I is also saturated, then increasing TAP would not increase lysis due to increasing antigen presentation on a single cell, as was seen in the RMA augmentation CTL assay. However, the peptide transfer assay suggests an alternative fate for peptides, which are presumably in excess of MHC class I requirements. If we take the model one step further, then excess peptides in the ER would be secreted where they can bind to neighbouring cells. More cells displaying antigen will result in more T lymphocytes being called into action. Therefore the immune response to an antigen is dependent on 117 the amount of antigen available, suggesting that the immune response could be increased simply by providing more TAP. This model is supported by the peptide transfer assay. One problem with subunit vaccines is the lack of crossreactive peptides which could immunize against all serotypes or be presented by different MHC alleles (167). We have shown that a peptide can be delivered to TAP in the form of a minigene in a viral vector. If multiple peptides were required they could be delivered as a gene encoding one protein containing multiple epitopes, as several minigenes encoding for multiple peptides in one virus, or as a mix of viruses containing various minigenes. If TAP were to be included in a T cell vaccine, its specificity is broad enough to accommodate multiple disparate peptides for the various immunodominant epitopes the different MHC alleles require. One limitation is that TAP would always have to be injected in some DNA form so it could undergo normal processing. Currently, the only two systems available are the attenuated or replication defective viral vectors, and the direct injection of DNA (148). 7.2 Tumour antigen presentation The effect of TAP on making cells susceptible to CTL mediated killing is not only relevant in situations where wild type levels of TAP are found but also in conditions where cells are deficient in TAP. The murine small cell lung carcinoma cell line, CMT.64, is deficient in MHC class I restricted antigen presentation (89,207), in which TAP, LMP 2 and 7, MHC class I heavy chain and (32M are all downregulated (89). However, it has been shown that the restoration of TAP expression in the cell 118 was sufficient to restore endogenous antigen presentation although not to wild type levels (89,120). The research in this thesis demonstrates that expressing TAP1 in CMT.64 restored antigen presentation sufficiently, in vivo, to improve survival of the mice inoculated with TAP-transfected challenge doses of CMT.64 and that the response was T cell mediated. Expression of both TAP subunits was not required for in vivo antigen presentation and recognition in TAP deficient cancer cells. Whether there is expression of TAP homodimers or cytokine induced expression of CMT.64's endogenous TAPs is not known. Further research has to be done to determine which of these models is true. It would also be interesting to determine which peptides the various CMT-TAP transfectants transport. This would provide additional information on the peptide specificities of the TAP complex, as well as help determine which protective peptides the MHC class I are presenting. This could indicate which immunodominant peptide provided protection in CMT 1.4, and whether the peptide was a tumour antigen, or a previously unseen peptide which the new rat TAP transported. Extraction of the peptides from MHC class I, and the ER lumens have been carried out in the Jefferies lab, but are awaiting sequence analysis. Knowledge of the protective peptide might also provide some insight into immunization with tumour antigens. An attempt was made to see if whole cell immunization could be achieved with the addition of TAP. CMT64, after transfection with TAP1 showed a delay in tumour growth, permitting longer survival times of the test mice. However, immunization with dead cells was not effective. The addition of TAP1 into CMT.64 was sufficient to 119 sensitize the cell to CTL lysis in the in vivo tumour experiment but it was not sufficient for turning the cell into a strong immunogen for priming tumour specific CTL. Poorly immunogenic tumour cells is a common phenomenon due to either inadequate antigen presentation or lack of the costimulatory signal required for T cell activation (231,232). The FACS, CTL and in vivo tumour data suggest that antigen presentation was enough to be recognized by a CTL. Therefore the poor immunization was more likely due to a lack of the costimulatory signal. A possible solution to making TAP transfected CMT.64 more immunogenic is to co-trans feet the cell with B7. B7 has been very effective in making many tumours more immunogenic. For example, B7 transfected melanomas were able to elicit a CD8 + anti-melanoma response when the untransfected melanoma could not (171). Even though we were unable to get a strong response from whole cell immunizations, some success was seen when TAP was included in a VV. The reason that the VV-TAP therapy worked better than the whole cells is unknown but there is one possible explanation. The VV vector itself could provide some T helper effects which may upregulate beneficial costimulatory molecules or increase cytokine production. This would increase lymphocyte activation and aid in tumour destruction. The viral vector containing TAP used in this research restored the ability of the tumour cell to display its own set of peptides which would be beneficial in a heterogeneous tumour population where all TAAs would be presented. Viral vectors, such as Adenovirus containing the MART-1 tumor antigen, have been successful in providing antitumour protection. But it still requires the knowledge of specific TAAs 120 and even then not all tumor antigens are successful in reversing the cancer due to the heterogeneous nature of neoplasms (197). There has also been the successful use of viral vectors containing the B7 costimulatory molecules, but again the presentation of the appropriate tumor antigen on the cancer cell is required which would not work for tumours with a MHC loss phenotype. Overall, the use of TAP in a viral vector could help overcome the difficulties in determining which peptides to present in a heterogeneous tumour population and could be used in conjunction with costimulatory molecules. One of the potential pitfalls in the use of TAP in vaccines and cancer therapeutics is the possibility of provoking an autoimmune reaction. The risk of autoimmunity would probably be low since the human TAPs are already quite promiscuous in terms of peptide selectivity. Although they limit the length of peptides transported they do not have a requirement for carboxy-terminal residues like the more stringent mouse TAP (96,98,99). Also, investigation into the influence of TAP polymorphism on peptide transport in mice and humans has demonstrated species specific transport patterns but no significant difference in selectivity by different TAP alleles within a species (233). It is therefore unlikely that any new peptides would be displayed. However, the full peptide repertoire of the TAP complexes has yet to be determined and the possibility of some allelic differences could arise. Recently it was determined in a peptide transport assay that a single point mutation in TAP2 could significantly alter the peptide transport pattern (221). Further research into the peptide specificity of various TAP alleles is needed to determine what effect the expression of TAP will have on antigen presentation. 121 8. Nomenclature ABC ATP-binding cassette APC antigen presenting cell ATCC American Type Culture Collection ATP adenosine triphosphate p2m P2-microglobulin P-ME P-mercaptoethanol BSA bovine serum albumin CMT.64 murine small cell lung cell line (TAP1 and 2 deficient) CMT.neo CMT.64 transfected with vector alone CMT1.4 CMT.64 transfected with rat TAP1 CMT1.10 CMT.64 transfected with rat TAP1 CMT2.1 CMT.64 transfected with rat TAP2 . CMT2.10 CMT.64 transfected with rat TAP2 CMT12.12 CMT.64 transfected with rat TAP1 and 2 CMT12.21 CMT.64 transfected with rat TAP1 and 2 Cr chromium CTL cytotoxic T lymphocyte DNA deoxyribonucleic acid DTT dithiothreitol EDTA ethylenediaminetetraacetic acid ER endoplasmic reticulum FACS fluorescence activated cell sorter FBS fetal bovine serum Hepes 4-(2-hydroxyethyl)-l-peperazineethanesulfonic acid HI heat inactivated HSP heat shock protein hTAP human TAP IFN interferon Ii invariant chain for MHC class II IR immune response ISCOMs immunostimulating complexes kbp kilobasepair kDa kilodalton LMP low molecular weight protein nxAb monoclonal antibody MDP muramyl dipeptide MHC major histocompatibility complex MOI multiplicity of infection MPL monophosphoryl lipid A mTAP murine TAP NK natural killer PAGE polyacrylamide gel electrophoresis 122 PBS phosphate buffered saline pCTL precursor CTL pfu plaque forming units P/S penicillin/streptomycin RNA ribonucleic acid rTAP rat TAP rVV recombinant Vaccinia virus SA-HRPO streptavidin-horse radish peroxidase SCLC small cell lung carcinoma SDS sodium dodecyl sulfate TAA tumour associated antigen tk thymidine kinase TAP transporter associated with antigen processing Th T helper lymphocyte VSV vesicular stomatitis virus VV Vaccinia virus VV-rTAPl Vaccinia virus containing the rat TAP-1 gene VV-hTAPl Vaccinia virus containing the human TAP-1 gene VV-hTAP2 Vaccinia virus containing the human TAP-2 gene VV-hTAPl-2 Vaccinia virus containing the human TAP-1 & 2 gene VV-NP Vaccinia virus containing the VSV N52-59 minigene VV-ESNP Vaccinia virus containing the VSV N52-59 minigene with the ER signal sequence in front WT wild type W/V weight per volume XMH xanthine, mycophenolic acid, hypoxanthine 123 9. 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