Towards Immunotherapy of Midgut Carcinoid Tumors

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 306

SOFIA VIKMAN

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List of papers

This thesis is based on the following papers, which will be referred to in the text by their roman numerals:

I Vikman S., Essand M., Cunningham JL., de la Torre M., Öberg K., Tötterman TH. and Giandomenico V. Gene Expression in Mid- gut Carcinoid Tumors: Potential Targets for Immunotherapy. Acta Oncologica 2005; 44(1):32-40.

II Essand M., Vikman S., Grawé J., Gedda L., Hellberg C., Öberg K., Tötterman TH. and Giandomenico V. Identification and Characterization of a Novel Splicing Variant of Vesicular Mono- amine Transporter 1. Journal of Molecular Endocrinology 2005; 35(3):489-501.

III Vikman S., Giandomenico V., Sommaggio R., Öberg K., Essand M. and Tötterman TH. CD8+ T cells against Multiple Tumor- associated Antigens in Peripheral Blood of Midgut Carcinoid Pa- tients. Cancer Immunology, Immunotherapy 2008; 57(3): 399-409.

IV Vikman S., Sommaggio R., de la Torre M., Öberg K., Essand M., Giandomenico V., Loskog A. and Tötterman TH. Midgut Carci- noid Patients Display Increased Numbers of Regulatory T cells in Peripheral Blood with Infiltration into Tumor Tissue. Manuscript.

Reprints were made with permission from the publishers

Contents

Introduction...... 13 The dispersed neuroendocrine system...... 13 General overview...... 13 Neuroendocrine cellular markers...... 13 Neuroendocrine cells of the gastrointestinal tract...... 14 Classical midgut carcinoid tumors...... 16 The immune system ...... 18 General overview...... 18 Antigen presentation...... 19 Dendritic cells...... 21 T cell activation and tolerance...... 23 Tumor immunology...... 26 History of tumor immunology...... 26 Immune recognition of tumors ...... 27 Immune escape mechanisms used by tumors ...... 28 Tumor antigens...... 30 Tumor antigen discovery ...... 31 T cell immunotherapy of tumors...... 32 General overview...... 32 Modified dendritic cell vaccines...... 32 Adoptive transfer of in vitro activated T cells ...... 32 Genetically engineered T cells...... 33 Present investigation ...... 34 General aim ...... 34 Specific aims ...... 34 Materials and methods ...... 34 Results and discussion ...... 35 Paper I: Gene Expression in Midgut Carcinoid Tumors: Potential Targets for Immunotherapy...... 35 Paper II: Identification and Characterization of a Novel Splicing Variant of Vesicular Monoamine Transporter 1 ...... 36 Paper III: CD8+ T cells against Multiple Tumor-associated Antigens in Peripheral Blood of Midgut Carcinoid Patients ...... 38

Paper IV: Midgut Carcinoid Patients Display Increased Numbers of Regulatory T cells in Peripheral Blood with Infiltration into Tumor Tissue...... 40 Conclusions...... 42 Acknowledgements...... 43 References...... 46

Abbreviations

aa Amino acid ADCC Antibody-dependent, cell-mediated cytotoxicity AINR Activation-induced, non-responsiveness APC Antigen presenting cell APUD Amine precursor uptake and decarboxylation CCL Chemokine (C-C) motif ligand CCR Chemokine (C-C) motif receptor CD Cluster of differentiation cDNA Complementary DNA CD40L CD40 ligand CDX-2 Caudal type homeobox transcription factor 2 CEA Carcinoembryonic antigen CFSE Carboxyfluorescein diacetate succinimidyl ester CGA Chromogranin A CML Chronic myeloid leukaemia CNS Central nervous system CpG Cytosine-phosphate-guanine CT Cancer-testis CT Computed tomography CTL Cytotoxic T lymphocytes CTLA-4 Cytotoxic T lymphocyte-associated antigen 4 DC Dendritic cell DNA Deoxyribonucleic acid dsRNA Double-stranded ribonucleic acid EBV Epstein-Barr virus EC Enterochromaffin EGFP Enhanced green fluorescent protein ER Endoplasmic reticulum ERAP1 Endoplasmic reticulum aminopeptidase 1 FLIP FLICE-inhibitory protein Foxp3 Forkhead box p3 GAPDH Glyceraldehyde-3-phosphate dehydrogenase GI Gastrointestinal GM-CSF Granulocyte macrophage colony stimulating factor HER-2 Human epidermal growth factor receptor 2 5-HIAA 5-Hydroxy indol acetic acid

HLA Human leukocyte antigen HPV Human Papilloma virus 5-HT 5-Hydroxytryptamine IA-2 Islet autoantigen-2 IDO Indoleamine-2,3-dioxygenase IFN Interferon IgG Immunoglobulin G IL Interleukin IPEX Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome LDCV Large dense core vesicles LFA Leukocyte function-associated antigen LPS Lipopolysaccharide M-CSF Macrophage colony stimulating factor mDC Myeloid dendritic cell MHC Major histocompatibility complex mRNA Messenger ribonucleic acid MRT Magnetic resonance tomography NCI National Cancer Institute NFAT Nuclear factor of activated T cells NFB Nuclear factor kappa B NK Natural killer PAMP Pathogen-associated molecular pattern pDC Plasmacytoid dendritic cell PET Positron emission tomography PGE2 Prostaglandin E2 PGP 9.5 Protein gene product 9.5 PI-9 Protein inhibitor 9 PRR Pattern recognition receptors RT-PCR Reverse transcription polymerase chain reaction SEREX Serological identification of antigens by recombinant expression cloning SLMV Synaptic-like microvesicles SNSD Self/Non-self discrimination TAP Transporter associated with antigen processing TCM Central memory T cells TCR T cell receptor TEM Effector memory T cells TGF Tumor growth factor TH T helper TH1 T helper type 1 TH2 T helper type 2 TH3 T helper type 3 TILs Tumor infiltrating lymphocytes

TLR Toll-like receptor TNF Tumor necrosis factor TPH Tryptophan hydroxylase TR1 Regulatory type 1 cells Tregs Regulatory T cells VAT Vesicular amine transporter VEGF Vascular endothelial growth factor VMAT Vesicular monoamine transporter WB Western blot WHO World Health Organisation

Introduction

The dispersed neuroendocrine system

General overview Neuroendocrine cells are distributed throughout most tissues in the body and they can be organized as macroscopic organs, such as the adenohypophysis and the adrenal medulla, or dispersed among other cell types as single or small groups of cells, such as the islets of Langerhans. The cells are specialized in the synthesis, storage and regulated secretion of hormones, bioactive peptides and amines important for body homeostasis1. Neuroendocrine cells were originally thought to be derived from neural crest cells as they expressed markers for neuronal differentiation and proteins involved in the biosynthesis of neurotransmitters. A.G. Pearse introduced the amine precursor uptake and decarboxylation (APUD) cell concept based on the ability of neuroendocrine cells to synthesize bioactive amines such as dopamine, adrenaline, noradrenaline, serotonin och histamine2,3. It has since been shown that, although sharing many common features with neurons, gastrointestinal neuroendocrine cells are of endodermal origin4-6. The following criteria are used to define a neuroendocrine cell; production of a neurotransmitter or a peptide hormone; presence of cytoplasmic dense core secretory granules, from which the content is released by regulated exocytosis, and the absence of axons and synapses7. Two regulated pathways of secretion are recognized in neuroendocrine cells: the amine/hormonal content is stored either in electron-dense granules called large dense core vesicles (LDCV) or in smaller synaptic-like microvesicles (SLMV) that are similar to the synaptic vesicles of nerve endings. The vesicular content is released in an endocrine or a paracrine mode in response to appropriate stimulation1,8.

Neuroendocrine cellular markers Neuroendocrine cells were originally identified due to their binding of chro- mium and silver salts, hence their former names chromaffin or argen-

13 taffin/argyrophil cells1. Presently, immunohistochemistry using specific protein markers are used to visualize these cells. Chromogranins are a family of acidic proteins stored in high concentra- tions in LDCVs of neuroendocrine cells and are therefore considered an important neuroendocrine immunocytochemical marker. The family includes chromogranin A, B and C. Chromogranins have several biological roles in neuroendocrine cells, chromogranin A (CGA) alone drives LDCV biogenesis and hormone sequestration in LDCVs. The protein is also processed into several biologically active peptides and secreted9,10. Synaptophysin is a synaptic vesicle protein involved in exo- and endocy- tosis in neuronal and neuroendocrine cells. Synaptophysin regulates, together with several other vesicle and membrane proteins, vesicle biogenesis, the formation of the fusion core complex, the fusion pore and initiate endo- cytosis11. Vesicular monoamine transporters (VMATs) are involved in the accumulation of amines in secretory vesicles. VMATs are integral proteins in the vesicular membranes of both neurons and neuroendocrine cells and use a proton gradient to translocate positively charged amines into secretory vesicles of the cells12. Somatostatin receptors are often expressed on cells of neuroendocrine origin. These cell surface receptors mediate inhibition of hormonal and amine secretion by neuroendocrine cells through binding of somatostatin13-15. The somatostatin receptors are often used in the clinic for somatostatin receptor scintigraphy, a well-established functional imaging technique for neuroendocrine tumors16.

Neuroendocrine cells of the gastrointestinal tract Neuroendocrine cells scattered within the gastrointestinal (GI) mucosa from stomach to colon represent the largest population of hormone-producing cells in the body17. There are at least 14 different neuroendocrine cell types in the GI tract, all with a specific regional distribution. The cells are specialized in the synthesis, storage and secretion of a wide range of polypeptide hormones and amines, all of which have an important role in bowel move- ment and fine tuning of hormonal secretion along the GI tract (Table 1). Enterochromaffin (EC) cells are widely distributed throughout the GI tract and specialize in the synthesis, storage and release of serotonin (5- hydroxytryptamine, 5-HT). Serotonin is synthesized from the amino acid tryptophan through a hydroxylation step involving the enzyme tryptophan hydroxylase (TPH) and a final decarboxylation step by an aromatic L-amino acid decarboxylase18 (Figure 1).

14 Table 1. Overview of GI neuroendocrine cells, modified from [8].

Vesicular content: Vesicular markers: Regional distribution:

Cell type Hormone/Peptide/Amine LDCV SLMV Pan Sto Duo Jej Ile App Col Rec

P/D1 Ghrelin CGA, VMAT2 Syn

EC Serotonin CGA, VMAT1 Syn

D Somatostatin CGA Syn

L Glucagon-like peptides CGA, CGC Syn

A Glucagon CGA, CGC, VMAT2 Syn

PP Pancreatic polypeptide CGA, CGC, VMAT2 Syn

B Insulin CGA, VMAT2 Syn

ECL Histamine CGA, VMAT2 Syn

G Gastrin CGA Syn

CCK Cholecystokinin

S Secretin, Serotonin CGA

GIP Gastric Inhibitory Peptide CGA

M Motilin

N Neurotensin CGA

LDCV=Large dense core vesicles, SLMV=Synaptic-like microvesicles, CGA=Chromogranin A, CGC=Chromogranin C, VMAT=Vesicular monoamine transporter, Syn=Synaptophysin, Pan=Pancreas, Sto=Stomach, Duo=Duodenum, Jej=Jejunum, Ile=Ileum, App=Appendix, Col=Colon, Rec=Rectum

Serotonin exerts critical functions within the central nervous system (CNS) and is involved in several neuropsychiatric disorders such as anxiety, depression and schizophre- nia19. However, EC cells ac- Gut lumen count for more than 90% of all serotonin synthesized in EC cell our body. Serotonin consti- Tryptophan tutes a key neurotransmitter TPH 5-Hydroxy within the enteric system with tryptophan important effects on intestinal AADC 5-HT motility and secretion. EC cells act as intestinal sensory 5-HT transducers that respond to mechanical pressure, chemical Enteric neuron stimuli and nutrients and the resulting serotonin release Figure 1. Synthesis and secretion of 5-HT initiates peristaltic and secre- by gut enterochromaffin cells.

15 tory reflexes by affecting enteric neurons, smooth muscle cells and entero- cytes20-22.

Classical midgut carcinoid tumors

Origin and classification Transformation of neuroendocrine cells gives rise to carcinoid tumors. The term “carcinoid”, meaning carcinoma-like, was coined in 1907 when Öber- endorfer described epithelial tumors in the gut that were morphologically distinct and less aggressive in behaviour compared to the well-known carci- nomas23. In 1963, Williams and Sandler divided carcinoids according to their em- bryological origin into foregut (lung, stomach, duodenum, upper jejunum and pancreas), midgut (lower jejunum, ileum, appendix and caecum) and hindgut carcinoids (colon and rectum)24. This classification was the first to emphasize clinicopathological differences between the groups but proved to be very imprecise since the tumors within each group varied greatly in mor- phology, function, biological behaviour and prognosis. In 2000 the World Health Organisation (WHO) published a new classification of carcinoid tumors using the terms neuroendocrine tumor and neuroendocrine carcinoma (Table 2). This classification takes into account localization and various morphological and biological criteria such as tumor size, angioinvasion, proliferative activity, histological differentiation, presence of metastases, invasion of adjacent organs and hormonal activity. The term carcinoid tumor has however not been abandoned and is often used synony- mously with the terms well-differentiated neuroendocrine tumor and carci- noma25,26.

Table 2. Classification of ileal neuroendocrine tumors, modified from [25]

1. Well-differentiated neuroendocrine tumor (carcinoid) Benign: Non-functioning or serotonin-producing, confined to the mucosa-submucosa, non-angioinvasive, 1cm. Benign or low-grade malignant: Non-functioning or serotonin-producing, confined to the mucosa-submucosa, angioinvasive, >1cm.

2. Well-differentiated neuroendocrine carcinoma (malignant carcinoid) Low-grade malignant: invasion of the muscularis propria or metastases, non-functioning or serotonin-producing with carcinoid syndrome

3. Poorly differentiated neuroendocrine carcinoma High grade malignant

16 Incidence and survival Overall carcinoid incidence rate is 2.5-5.2 cases per 100,000 per year27-29. Carcinoids occur most frequently in the gastrointestinal tract (66%), with the bronchopulmonary system as the second most common location (31%). The midgut carcinoids constitute approximately 30% of all carcinoids28,30,31. Ac- cording to SEER, a National Cancer Institute (NCI) registry, the five-year survival rates for midgut carcinoid patients between the years 1992-1999 ranged from 64% for patients with localized disease to 44% for patients with distant metastases28. The age-adjusted, overall 5-year survival rate for patients with jejunal/ileal carcinoids in Sweden between the years 1960-2000 was 56% according to a report based on the Swedish National Cancer Regis- try. Presence of liver metastases and carcinoid heart disease were described as the most significant adverse prognostic factors32.

The carcinoid syndrome Midgut carcinoid tumors grow slowly and unperceived and often do not become clinically evident before the appearance of hepatic metastases causing a series of symptoms referred to as “the carcinoid syndrome”. These symptoms include diarrhoea, flushing, bronchial constriction and right-sided valvular heart disease33. Heart valve fibrosis and diarrhoea have been attrib- uted the high plasma levels of serotonin due to the excessive secretion by the tumor cells and tachykinins have been implicated in the cause of flush34-36. Symptoms of the carcinoid syndrome is present in approximately 70% of patients diagnosed with midgut carcinoid and about 30% of the patients display symptoms of carcinoid heart disease37. Patients may also experience abdominal pain and intestinal obstruction due to the excessive fibrosis associated with the tumor38. Non-functioning or small primary tumors are often incidental findings at surgery39.

Diagnosis Diagnosis of midgut carcinoids is primarily based on histopathology and positive staining for serotonin and the neuroendocrine vesicular markers CGA and synaptophysin. Biochemical markers such as elevated urinary levels of the serotonin metabolite 5-hydroxy-indol-acetic-acid (5-HIAA) and serum CGA are also used for diagnosis and disease monitoring39,40. Plasma levels of CGA have been shown to correlate with tumor burden41-43 and have proven a valuable biochemical marker for tumor growth and treatment response in patients with limited disease44. Different radiological techniques such as computed tomography (CT), magnetic resonance tomography (MRT), somatostatin receptor scintigraphy and positron emission tomography (PET) are valuable methods for the detection and monitoring of metastatic spread16,45.

17 Current treatment Current treatments of metastasized midgut carcinoids aim at controlling tumor growth and hormonal secretion. Palliative surgery can reduce the tumor burden and is commonly combined with medical treatment. Cytostatic drugs are of limited use due to the generally low proliferation rates for midgut carcinoids. Instead, medical treatment involves administration of interferon (IFN) and somatostatin analogues, such as octreotide and lanreotide, which efficiently relieve symptoms of the carcinoid syndrome by inhibiting hormonal secretion. Somatostatin analogues have also been described to induce disease stabilization in approximately half of treated patients, mainly through induction of apoptosis and inhibition of angiogenesis. Actual tumor reduction has however only been described in a minority of patients, about 5%. IFN may also induce disease stabilization by inhibiting tumor cell growth and angiogenesis, but significant anti-tumor effects have not yet been described39,46-48. Experimental trials using radioactively labelled somatostatin analogues targeting tumor cells have been performed with some tumor re- duction49.

The immune system

General overview The immune system is a highly complex network of cells whose primary function is to protect our body from harm. An essential task for the immune system is to detect and react against dangerous substances such as patho- genic microorganisms, toxins and malignant cells that threaten the integrity of our tissues. At the same time the immune system must be able to accept and create tolerance to foreign entities that do not cause harm, like foetuses, commensal bacteria, ingested food and inhalated pollen. In addition, whenever encountered with a potential threat the immune system must determine in cooperation with the affected tissue how and what type of immune response should proceed in order to clear the danger50,51. Our immune system can be divided into an early, non-specific innate entity that provides a first line defence against potential danger, and a later, more specific, adaptive response which follows an innate immune activation. Innate immunity includes physical barriers with acidic and antimicrobial properties provided by our skin and mucosa, phagocytic cells such as macrophages, dendritic cells (DCs), eosinophils and basophils, other haema- topoietic cells such as natural killer (NK) cells, mast cells and T cells. The complement system, acute phase proteins and inflammatory mediators are also important52. Adaptive immunity is mediated by lymphocytes; B cells

18 constitute the humoral branch with the production of antibodies which respond to soluble antigens. T cells form the cellular branch which responds to processed peptide antigens presented in the context of major histocompatibility molecules (MHC). Diversity, specificity and memory are cardinal features of adaptive immunity53-55.

Antigen presentation

Antigen presenting cells Antigen presenting cells (APC) are cells specialized in the capture and processing of antigens. APCs display processed antigens in the form of short peptides on their MHC class I and II molecules to T cells and provide the T cells with co-stimulatory signals and cytokines necessary for their proliferation and further effector differentiation. DC, B cells and macrophages can all function as specialized APCs, these cells are able to capture exogenous antigen from the extracellular environment on their MHC II molecules and pre- + 56,57 + sent them to CD4 T helper (TH) cells . The CD4 TH cells are the primary effector cells of adaptive immunity and important for further development of the immune response either into antibody production by differentiated B + cells (TH type 2 response) or to provide help for cytotoxic CD8 T cells 58 (CTL) (TH type 1 response) . Antibody-coated cells are targeted for destruc- tion by complement activation or by a process called antibody-dependent cell-mediated cytotoxicity (ADCC)59. CTLs kill target cells either by the release of cytotoxic proteins such as perforin and granzymes or by the interaction of Fas ligand with Fas receptors on the surface of the target cell. Both pathways result in apoptosis of the target cell. Virtually all nucleated cells in the body present endogenously synthesized proteins on their MHC I and are potential targets for killing by activated CTLs60. DCs are often referred to as professional APCs. They are the main APC that are able to process and transfer exogenous antigens onto their MHC I pathway, a phenomenon termed cross-presentation. This process is of particular importance for DCs to present exogenous antigens from a third affected cell to naïve CD8+ T cells. Due to their high capacity of providing co- stimulation DCs are excellent stimulators of naïve CD4+ and CD8+ T cells57.

Antigen processing pathways MHC molecules are highly polymorphic proteins that present peptide fragments of proteins from the intracellular and extracellular environment to T cells. They were initially recognized for their role in rejection of transplanted tissue, but have since been crystallized and analyzed in detail. Class I and class II MHC molecules, in humans denoted human leukocyte antigens (HLA), share both similarities and differences in their structure. Both classes consist of an extracellular domain containing the peptide-binding cleft fol-

19 lowed by immunoglobulin-like domains that, in addition to anchoring the molecule to the cell membrane, provide binding sites for the T cell co- receptors CD4+ and CD8+. Antigens from the intracellular and extracellular environment are processed and presented on the cell surface by MHC class I and II using different pathways (Figure 2)61.

MHC class I pathway All nucleated cells present processed intracellular proteins through the MHC class I pathway (Figure 2a). Ubiquitinated endogenous proteins, as well as proteins from viral and intracellular pathogens, are degraded in proteasomes, which are large cytosolic enzyme complexes with a broad range of prote- olytic activities, to generate peptide fragments up to 20 amino acids (aa) long. The generated peptides are translocated into the endoplasmic reticulum (ER) by a specialized transporter associated with antigen processing (TAP) and their N-terminals are further trimmed by the ER-aminopeptidase 1 (ERAP1). The resulting peptides of 8-10 aa are loaded onto MHC class I molecules and transported from the ER to the cell surface for presentation to CD8+ T cells57,61,62.

MHC class II pathway Exogenous antigens captured from the extracellular environment are en- gulfed by specialized APCs and degraded enzymatically in endosomes to generate peptides of typically 12-18 aa. These endosomes are later fused with MHC II-rich exocytic vesicles from the ER. The MHC II peptide binding cleft is initially occupied by a blocking invariant chain peptide which is removed when the two compartments are fused and the processed exogenous peptides are loaded onto the MHC II (Figure 2b). The MHC II-peptide complexes are then transported to the cell surface for presentation to CD4+ T cells57,61.

Cross-presentation DCs, capable of cross-presentation, are able to transfer exogenously derived antigens onto their MHC I pathway by fusing phagosomes with ER-derived vesicles (Figure 2c). These hybrid vesicles contain newly synthesized MHC I molecules and components for peptide loading including TAP. Antigens can become cross-presented either independently of TAP by loading phagosome-degraded peptides directly onto MHC I molecules or the phago- cytosed antigens can be translocated out into the cytosol, degraded in closely associated proteasomes and transported back into the phagosome or the ER by TAP. The peptides are then loaded onto the MHC I molecules and transported to the cell surface for presentation to CD8+ T cells61,63,64.

20 a) b) c)

+ + CD8 T cell CD4+ T cell CD8 T cell

MHC I MHC I +peptide MHC II +peptide +peptide Exocytic Exogenous vesicle protein MHC II Endogenous protein MHC I Phagosome Exogenous Exocytic protein Proteasome MHC I vesicle Proteasome TAP

Exocytic MHC II MHC I vesicle Phagosome

TAP Late endosome MHC I MHC II Invariant MHC I chain ER ER ER

Figure 2. Antigen processing pathways for T cell presentation. a) The MHC I pathway of endogenous antigens for CD8+ T cell presentation, b) The MHC II pathway of exogenous antigens for CD4+ T cell presentation and c) Cross- presentation of exogenous antigens to CD8+ T cells.

Dendritic cells

Origin and subsets DCs differentiate from CD34+ bone marrow-derived stem cells. Human DCs are divided into two main populations: myeloid and plasmacytoid DCs. Myeloid DCs (mDCs) have a monocytoid appearance, are thought to differentiate from myeloid progenitors and are potent activators of T cells. Plas- macytoid DCs (pDC) have the appearance of plasmablasts, are thought to differentiate from lymphoid progenitors and are the main producers of type I IFNs in response to viral infections. pDCs have a more limited T cell priming capacity and have been described as an “innate DC” that provides a link between innate and adaptive immune responses65-67.

Maturation status of the DC regulates tolerance and immunity DCs are considered key players in the regulation of immune responses. The decision between immunity and tolerance in response to an antigen is made by DCs depending on their maturation status. DCs are in their immature state mainly localized in peripheral tissues and specialize in the uptake and proc-

21 essing of antigens. They circulate through local draining lymph nodes where their antigenic content is presented to T cells. If no peripheral danger is present the stimulatory capacity of the DC is low and the outcome of T cell recognition will be induction of tolerance. In contrast, whenever the DC encounters danger signals due to tissue distress, for example following injury or the invasion of microbes, the DC rapidly differentiates into a mature phenotype with high T cell stimulatory capacity55,68,69. Several maturating stimuli for DCs have been described; bacterial products such as lipopolysaccharide (LPS) and flagellin, double-stranded ribonucleic acid (dsRNA), cytosine-phosphate-guanine deoxyribonucleic acid (CpG DNA), heat-shock proteins, breakdown products of hyaluron and inflammatory mediators like tumor necrosis factor (TNF) and interleukin 1 (IL-1) are a few examples70,71. Many of these mediators bind to specific Toll- like receptors (TLR) present on the DC which initiate the maturation proc- ess72. DC maturation results in chemokine (C-C motif) receptor Dendritic cell T cell 7 (CCR7) expression CD40 CD40L PD-1 and mature DCs home PD-L1, PD-L2 ICOS-L ICOS to lymph nodes where CD80,CD86 CD28,CTLA-4 CCR7 ligands are MHC I/II CD8/CD4 with peptide TCR present at high con- CD58 (LFA-3) CD2 centrations. DC matu- CD54 (ICAM-1) CD11a (LFA-1) ration also results in enhanced presentation of the antigenic content. Mature DCs Figure 3. T cell priming by DCs. The immunologi- upregulate the cell cal synapse between DCs and T cells involves a surface expression of multitude of molecular interactions. The outcome of this interaction can be either activation or tolerance. MHC I and II molecules and the expression of CD80 (B7.1) and CD86 (B7.2), co-stimulatory molecules essential for T cell activation. The co-stimulatory receptor CD40 is also upregulated + for interaction with CD40 ligand (CD40L) on activated CD4 TH cells, which is important for further differentiation and augmentation of the immune response. Increased expression of adhesion molecules, such as CD54 and leukocyte function-associated antigen (LFA), is necessary for more sta- ble interactions with T cells (Figure 3)73. The interaction between DCs and T cells mediate reciprocal communica- tion, DCs activate or tolerize T cells and T cells can influence the activation status of the DC. CD40L is a potent enhancer of stimulatory capacity, while cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is an important negative regulator. CTLA-4 signalling efficiently inhibits T cell proliferation

22 and induces indoleamine-2,3-dioxygenase (IDO) expression in DCs. IDO is a catabolic enzyme that degrades the essential amino acid tryptophan to kynurenins, which suppress T cell activation and proliferation74-76. DCs and other APCs are also key determinants of TH polarization. De- pending on the DC subset, its micromilieu and Dendritic cell/APC the type of stimulus inducing DC maturation, the TH cells are polarized TH into a TH1 or a TH2 re- IL-12 IL-4 sponse (Figure 4). Vi- ruses and intracellular IFN CTL TH 1 TH 2 bacteria or parasites IL-4 induce strong innate IL-12 IL-10 IL-4 IFN IL-5 B immune activation, in- IL-2 IL-10 IL-13 cluding macrophages and NK cells, trigger IL- 12 production and TH1 polarization. Helminths and allergens do not elicit strong innate im- Figure 4. TH polarization. DCs direct the munity and no IL-12 and immune response towards TH1 or TH2. consequently promote TH2 polarization. Each subset amplifies itself and cross-inhibit the other, consequently once an immune response develops along one pathway it be- comes increasingly polarized in that direction77,78.

T cell activation and tolerance

Cytotoxic T cell activation requires T cell help A sufficient and lasting CTL response requires presentation of antigen by + fully mature DCs and the help from activated CD4 TH1 cells. Mature DCs become licenced and acquire full CTL priming capability by activated TH1 cells79. A DC that encounters danger signals from its surroundings presents its antigenic content on MHC II together with co-stimulation to activate CD4+ + TH cells. The CD4 TH cell receives a signal through its T cell receptor (TCR) (Signal 1) and a second co-stimulatory signal through CD28 (Signal 2) and this leads to an upregulation of CD40L. The CD40-CD40L interaction provides the DC with the ultimate maturation stimuli to further promote and amplify a cytotoxic TH1-response. The licensed DC now has full co- stimulatory properties and produces large amounts of IL-12, a cytokine piv-

23 otal for CTL activation and effector function and sometimes referred to as Signal 3. Naïve CD8+ T cells recognizing (cross) presented antigens on MHC I of the DC receive signal 1 through their TCRs, signal 2 through CD28 and signal 3 by IL-12. The cells become activated, differentiate into CTLs and go through clonal expansion (Figure 5). The activated TH1-cell is thus not only important for licensing the DC; it is also indispensable in providing help in the form of large amounts of IL-2 and IFN to drive CTL proliferation. Failure in providing the CTLs with proper co-stimulation and helper cytokines result in anergy80-83.

IL-12 (Signal 3)

MHC I TCR + CTL Peptide CD8 CD8 T cell CTL Mature DC Signal 1

CTL Signal 2 CD28 CD80/CD86

+ CD40 Effector CD8 cells MHC II CD80/CD86 Peptide IL-2, IFN

TCR CD4 CD40L upregulation CD28

Signal 1

IL-12 Signal 2 Th1 cell

Effector CD4+ cell

Figure 5. CTL activation. A mature DC licensed by an activated TH1 cell ac- quires full CTL priming capacity. Activated CD8+ T cells go through clonal expansion with the help of cytokines produced by the TH1 cells.

Memory T cells Immunological memory is characterized by the ability of experienced lymphocytes to respond better and more rapidly upon secondary encounter with the same antigen. Memory CD4+ and CD8+ T cells are divided into two subsets; T central memory (TCM) and T effector memory (TEM) cells. TEM are found within tissues and are able to carry out immediate effector functions

24 while TCM cells express lymph node homing molecules, such as CD62 ligand and CCR7, and are thought to mediate more long term memory. TCM cells are able to rapidly proliferate and secrete IL-2 in response to antigen stimulation and differentiate into effector cells that are able to produce IFN. Generation of memory CD8+ T cells requires optimal CTL activation through CD4+ T cell help. During the end of the effector phase a majority of effector CTLs die by apoptosis, however, a small fraction of 5-10% of effector cells survive, differentiate and enter the long-lived memory CD8+ T cell pool. The cytokine IL-15 has been shown to be important for homeostatic 84-87 proliferation of memory cells and IL-7 appears important for survival .

Peripheral tolerance of cytotoxic T cells Apart from central tolerance generated in the thymus there are a number of peripheral mechanisms that protect us from harmful autoimmunity caused by self-reactive T cells. Peripheral tolerance of CD8+ T cells can occur either through anergy or clonal deletion. CD8+ T cells can also be actively sup- pressed by regulatory T cells (Tregs). Any naïve T cell receiving TCR en- gagement (signal 1) in the absence of co-stimulation are rendered anergic, a state characterized by functional unresponsiveness with inhibited cytokine production and limited proliferation. This state can only be reversed in the absence of antigen88. When the antigen persists and the TCR interaction is strong the state of anergy is maintained in the CD8+ T cell. On the other hand, if the antigen persists and the TCR interaction is weak the outcome is more likely clonal deletion. Anergy is thus an active process dependent on continuous presence and strong recognition of antigen89. When provided with co-stimulation (signal 1 and 2) by a fully mature DC a naïve CD8+ T cell can undergo autocrine IL-2 dependent clonal expansion, but within 3-4 days it loses its ability to produce IL-2 and reaches a state called activation-induced non-responsiveness (AINR). The effector functions are retained but proliferation ceases unless IL-2 is provided through CD4+ helper cells. The AINR state constitutes a regulatory checkpoint where activated T helper cells give the CTL response permission to proceed. Following reversal of AINR the CTLs are much less dependent upon co-stimulation and the ability to produce IL-2 is regained90. Effector CTLs are thought to be less susceptible to tolerance but memory CD8+ cells can again be tolerized if subjected to high doses of persistent antigen91.

Regulatory T cells Tregs are important for maintaining peripheral tolerance and protect us from destructive autoimmunity. In humans CD4+ Treg subsets include naturally occurring thymic-derived CD4+CD25+Foxp3+ Tregs and peripherally induced T regulatory type 1 (TR1)- and T helper type 3 (TH3) cells. Tregs are functionally defined as T cells that inhibit other immune cell functions either

25 directly through a cell-to-cell dependent contact or indirectly through the secretion of inhibitory cytokines, such as IL-10 and tumor growth factor (TGF). Tregs also express CTLA-4 constitutively. Tregs proliferate in response to a specific antigen, however, the immunosuppressive function is exerted in a non-antigen dependent fashion92,93. Immature DCs have been implicated as having a role not only to induce T cell anergy in the periphery, but also in inducing Tregs. Immature DCs, or suppressive DCs that have been exposed to IL-10, TGF, or inhibitory stimulation by CTLA-4 may induce Tregs that actively down-regulate effector responses94-97. The most specific marker for Tregs to date has been the forkhead box p3 (Foxp3) transcription factor, a member of the fox (forkhead/winged-helix) family of transcription factors. Evidence for the crucial role of Foxp3 in the development and function of Tregs came initially from studies in mice98,99, but has proven to be of similar importance in humans100. Foxp3 is a transcriptional repressor that inhibits the activity of nuclear factor of activated T cells (NFAT) and nuclear factor kappa B (NFB), two important transcription factors regulating genes involved in cytokine production and T cell function101. Mutations of the Foxp3 gene lead to severe autoimmunity, in humans known as the immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome102,103.

Tumor immunology

History of tumor immunology F.M. Burnet developed the self/non-self discrimination (SNSD) model in the 1950’s in which he stated that the immune system is able to discriminate between self and anything foreign to which it is activated against and should destroy104. On the basis of this model L. Thomas along with Burnet proposed the cancer immunosurveillance theory in which the immune system is described to continuously survey the body for developing neoplasia and will eliminate tumors as they arise105. This theory relied on the assumption that unique antigens expressed by the tumor would be regarded as non-self and the default reaction of a mature lymphocyte to a foreign antigen was activation. Important modifications to the SNSD model and the immunosurveillance hypothesis were provided by C. Janeway in the 1980’s. He postulated that the default reaction of the immune system is tolerance based on the Lafferty/Cunningham’s Two Signal Model106, where an APC is required to provide a co-stimulatory signal to the lymphocyte in order to activate it. Janeway also introduced pattern recognition receptors (PRR) expressed by the APCs that recognize pathogen associated molecular patterns (PAMP) of

26 molecules found on evolutionarily distant organisms like bacteria. Binding of PAMPs to the PRRs on the APC causes an upregulation of the ability to provide the co-stimulation necessary for an immune response initiation. Janeway thereby introduced the infectious non-self/noninfectious-self model that postulated the outcome of an immune response against tumors to be tolerance107,108. Despite these important modifications Janeway’s model could still not explain all aspects of tumor immunology. In the 1990’s P. Matzinger proposed yet another model for initiation of immune reponses namely the danger model. The danger model is based on the idea that the ultimate controlling signals for initiating an immune response are endogenous, not exogenous, and stem from the tissue where the APCs reside. The activation state of the APC and thereby its co-stimulatory ability is regulated by alarm signals from cells that are abnormally distressed or necrotically destroyed. PRRs on APCs also bind endogenous stress proteins like heat shock proteins in addition to molecules from exogenous pathogens and binding causes APC maturation. In contrast, cells that die by a normal physiological programmed cell death, apoptosis, do not send out danger signals. They become phagocy- tosed by local APCs and presented to T cells without co-stimulation and the outcome will be tolerance109. The danger model predicts that the default reaction of a tumor-reactive lymphocyte is inactivation and a danger signal is required to initiate an anti-tumor response. Since many tumors at least initially grow without causing any tissue damage and are essentially healthy cells, APCs in the tumor area are not activated and potentially tumor- reactive T lymphocytes are not provided with the necessary co-stimulation for activation. During progressive growth and metastasis the tumor may cause more tissue distress due to, for example, hypoxia and cell necrosis. This will alert local DCs to initiate an immune response against tumor antigens. But the response will only proceed for as long as danger signals are present, effector CTLs have a limited life-span and memory T cells are susceptible to tolerance induction by the continuous encounter with tumor antigens presented by tumor cells. The inability of APCs to become activated in the tumor area and the constant tolerization of T cells by tumor cells unable to provide signal 2 seem to be principal mechanisms of the failure of the immune system to completely eradicate tumors110.

Immune recognition of tumors Spontaneous immunogenicity of tumors within a host was for a long time a matter of debate. Growing evidence shows that many tumors are not ignored by the immune system, on the contrary, a tumor with progressive growth is often recognized by immune cells but rather than being a passive target it deviates the immune system to create a safe tolerogenic environment111.

27 Numerous reports about spontaneous tumor regressions strongly indicate that tumors can be recognized by the immune system. Spontaneous regressions are often connected to tissue distress such as bacterial infections or surgical trauma. However, regressions seldom lead to a complete cure, metastases continue to develop suggesting that the immune response once initi- ated has ceased112-114. Reports about paraneoplastic syndromes in patients that have been diagnosed with cancer after initially showing neurological symptoms also suggest that the immune system spontaneously has become activated by the tumor and unintentionally cause autoimmune damage on cross-reactive healthy tissue during the immune response115. The tumor area often shows infiltration of lymphocytes (TILs). Signifi- cant tumor regressions have been seen in adoptive cell transfer experiments where TILs have been isolated from tumor lesions, expanded ex vivo and infused back into the autologous patient. The results suggest that TILs are able to recognize tumor antigens, however, they seem unable to kill tumor cells in vivo116. Screening of tumor expression libraries with CTL clones derived from TILs has also been a successful method to identify tumor antigens, adding further strength to the fact that TILs recognize tumor117. Sero- logical identification of antigens by recombinant expression cloning (SEREX) is a different technique used to identify tumor antigens. Serum from cancer patients are screened for antibodies against tumor proteins and a humoral response is often seen in investigated patients118,119. The development of tetramers, MHC molecules with bound peptides cou- pled to a fluorescent dye, has provided an invaluable tool to investigate T cells with certain TCR specificity. By using tetramers with peptides derived from tumor antigens, cancer patients have been shown to harbour significantly higher number of T cells recognizing tumor antigen in their blood compared to healthy controls. This suggests that an immune activation against tumor antigens has occurred. Phenotypic and functional analyses of these T cells have shown that although displaying effector markers they were functionally impaired111,120,121.

Immune escape mechanisms used by tumors Several factors contribute to the tolerogenic microenvironment in which a tumor is allowed to grow. The tumor mainly expresses normal genes which our T cell repertoire has been shaped to accept through central mechanisms, i.e. thymic deletion and different peripheral mechanisms such as anergy, deletion and suppression by Tregs. T cells even capable of recognizing the tumor might therefore be comparatively scarce122. The tumor progresses relatively slow, often during a time course of several years, displaying many similarities with a chronic infection. The tumor microenvironment is often rich in factors that inhibit DC differentiation, maturation and function such as IL-10, TGF, macrophage colony stimulat-

28 ing factor (M-CSF), IL-6, IDO, vascular endothelial growth factor (VEGF), prostaglandin E2 (PGE2) and gangliosides. The lack of strong danger signals in the tumor area during progression causes local dendritic cells to remain in an immature state and sufficient CTL activation against acquired tumor antigens does not occur110,123. Tumors may also grow in a cytokine environment strongly shifted towards TH2 or TR1/TH3, a milieu favourable for the humoral entity of the adaptive immune system and suppressive of the cellular branch, respectively. An efficient cytotoxic immune response requires a shift towards a proin- flammatory TH1 milieu for tumor elimination. Expression of TH1 cytokines, such as granulocyte macrophage colony stimulating factor (GM-CSF), IL- 12, IFN is usually rare in the tumor microenvironment. TH2-dominance might be determined by the tissue, the gut is for example a typical TH2- dominated environment to prevent us from having undesired inflammatory reactions to ingested food and commensal bacteria. Tumor cells can also produce immunosuppressive factors, such as IL-10 and TGF or induce the production of these cytokines from associated macrophages and surrounding stroma in order to prevent a shift towards TH1. The consequential TR1/TH3 dominated environment results in quiescent DCs, induction of Tregs and anergic effector T cells123. Tregs and their contribution to tumor tolerance is a rapidly evolving field of investigation. Cancer patients are often reported as having increased numbers of Tregs both in the tumor area, metastastic lymph nodes as well as in peripheral blood124-127. Evidence that Tregs contribute to tumor tolerance has been shown in mice studies in which Tregs were eliminated by blocking antibodies against CD25+ 128. In humans, targeting Tregs by CTLA-4 specific antibody129,130, low-dose cyclophosphamide131 and the agent denileukin diftitox132,133 has in several studies improved anti-tumor therapy. Increased Treg infiltration in the tumor area also predicts poor survival in human cancer patients127. The chemokine (C-C motif) ligand 22 (CCL22) in the tumor area has also been implicated in the accumulation of Tregs in the tumor microenvironment. CCL22 stimulates Treg trafficking to the tumor by interaction with CCR4 on Tregs. Tregs have also been shown to specifically recognize tumor antigen134. Due to their genetically unstable nature, tumor cells have also acquired a number of escape mechanisms to avoid direct recognition and killing by immune cells. Down-regulation of MHC I expression is an excellent way of hiding from T cell recognition, defects in antigen processing and presentation such as mutations in TAP or proteosomal subunits as well135. Resistance to killing can also be achieved through defective apoptosis pathways and expression of apoptosis inhibitors such as protein inhibitor 9 (PI-9)136 and FLICE inhibitory protein (FLIP)137.

29 Tumor antigens

Cancer-testis antigens (Shared tumor antigens) Cancer-testis (CT) antigens are expressed by many different cancers of various histological origins. Expression in normal tissues is by definition restricted to germ cells of testis and occasionally ovary. CT antigens are often present as multigene families and frequently map to the X chromosome. The reason for CT antigen expression during both spermatogenesis and tumori- genesis can at least partly be explained by the DNA methylation status in these cells. Germ cells possess much lower levels of CpG methylated DNA compared to somatic tissues and cancer cells tend to exhibit a general ge- nome-wide demethylation. Upregulation of CT antigen expression can also be induced using a DNA demethylating agent. As a consequence there is a tendency of clustering of several CT antigens in a CT antigen positive tumor specimen while other tumor specimens are completely negative. Expression also seems to be associated with tumor progression and malignancy; several reports indicate higher expression of CT antigens in metastatic lesions compared to primary tumors. The cellular functions of CT antigens are with a few exceptions unknown although there are indications of putative roles in transcriptional regulation for several of them. The MAGE and GAGE/PAGE/XAGE gene families are examples of CT antigens widely expressed by many different cancers and NY-ESO being one of the most immunogenic CT antigen described. Many CT associated genes have been identified as well, they have a tissue expression with a cancer-testis profile: however no epitopes recognized by T cells have been identified yet and they are therefore not proven antigens138-140.

Differentiation antigens Differentiation antigens are lineage-specific antigens that are expressed by the tumor cells as well as the normal counterpart from which the tumor is derived. Targeting this type of antigen will result in an immune activation also against normal tissues expressing the antigen. However, if the affected normal tissue is considered non-essential for life or have a high regeneration rate this might not pose a severe problem. Malignant melanomas express some of the most well-characterized differentiation antigens: MART- 1/Melan A, gp100 and tyrosinase. These antigens are involved in the process of melanin production and are consequently also expressed by normal melanocytes138,140. Melanoma differentiation antigens have been successfully used in immunotherapy and have been shown to cause an immune attack on the tumor as well as vitiligo116.

30 Over-expressed antigens Genes ubiquitously expressed in various normal tissues can become overexpressed in tumor tissue and trigger T cell activation. The level of antigen expression in normal cells might be too low for breaking tolerance to it, but when the expression increases in a tumor cell it can give rise to an immunological response. Examples of overexpressed antigens are human epidermal growth factor receptor 2 (HER2), carcinoembryonic antigen (CEA), PRAME and SART138,140.

Tumor-specific antigens Antigens that are unique and exclusively expressed by a tumor can be considered truly tumor specific. They are commonly due to specific genetic alterations and often relate to tumor transformation. Examples include specific mutations of p53 and catenin140. Another well-known example is the bcr/abl fusion gene product involved in chronic myeloid leukaemia (CML), which has been shown to be recognized by T cells141.

Tumor-associated viral antigens The Epstein-Barr virus (EBV)142 and the human papilloma virus (HPV)143 are two viruses directly associated with cancer development. Tumor- associated viral proteins constitute excellent tumor antigens due to the strong antigenicity of viruses.

Tumor antigen discovery A number of methods have been used in the search for specific tumor antigens, several of which have identified gene products specifically expressed in tumor tissue144,145. It is however important to note that a protein specifically expressed in tumor tissue is not a proven antigen until it has been shown to be recognized by the immune system. Several tumor antigens, for example the first cancer-testis antigen MAGE-1, were originally identified through CTL epitope cloning. Many melanoma antigens in particular were identified by this method since it has been fairly easy to establish melanoma cell lines from patient tumor material. Patient T cells were tested for reactivity against autologous tumor cells and autologous tumor complementary DNA (cDNA) libraries to identify the relevant antigen146. SEREX exploits the B cell repertoire against the tumor in cancer patients and has lead to the identification of several tumor antigens. By screening tumor-derived expression libraries with patient sera tumor-associated immunoglobulin G (IgG) antibodies can be detected. A high titer IgG response in vivo indicates TH cell activation and given the fact that immune recognition of tumors is considered a concerted action may reflect that TH and CTL epi-

31 topes against the relevant antigen exist. SEREX has verified the existence of a humoral response against antigens originally described as CTL targets such as MAGEs but has also identified new CTL antigens such as the CT antigen NY-ESO1147,148.

T cell immunotherapy of tumors

General overview The aim of T cell immunotherapy of tumors is to trigger or enhance an immune response against tumor-associated antigens. Generation of an effective CTL response that selectively targets and eliminates tumor cells with as little cross-reactivity with normal tissue as possible is desired. Several approaches have been attempted experimentally and in the clinic.

Modified dendritic cell vaccines DCs modified in vitro have been used in several cancer trials to date. The aim is to generate or facilitate tumor-reactive TH1 immune responses in vivo. DCs, playing a central role in the initiation, coordination and regulation of immune responses, have been utilized to present tumor material in a variety of forms to the immune system. The maturation status and the costimulatory capacity of the DCs have received increasing attention since it has become apparent that these factors influence the quality of T cell activation149. Most commonly DCs have been pulsed with tumor lysate150,151 or tumor antigen- derived peptides152. DCs have also been fused with tumor cells to generate hybrids capable of initiating CTL activation combined with the endogenous expression of several tumor antigens153. DCs have also been transfected with RNA encoding tumor antigens154, transduced with viral vectors encoding tumor antigens155, cytokines156, chemokines157, and immunostimulatory molecules158. Trials using autologous DCs exposed to a recombinant fusion protein of a prostate cancer antigen and a DC targeting molecule (Provenge®), have recently been performed with promising results regarding survival advantage159-161. However, the clinical efficacy of DC vaccinations has in general been limited and there is an increasing awareness that DC vaccinations must be combined with other therapies targeting the tumor- induced immunosuppression in cancer patients162.

Adoptive transfer of in vitro activated T cells Clinical trials involving adoptively transferred anti-tumor CTLs have generated hope for patients with advanced malignant melanoma. Several trials

32 using TILs have resulted in anti-tumor responses and mediated tumor regression in patients. Tumor-induced suppression in the host can be circumvented by isolating T cells from tumor lesions, expanding tumor-reactive clones ex vivo and infusing these cells back to the patient. Tumor-reactive clones are identified in vitro based on cytokine production in response to tumor cells and amplified in the presence of allogeneic stimulators and IL-2. High doses of IL-2 are also given to the patients to improve survival of the infused T cells, although it is associated with high toxicity. In the case of melanoma, anti-tumor effects are often accompanied by vitiligo, i.e. autoimmune de- struction of normal melanocytes in the skin116,163-165. To improve therapy efficiency patients have been pre-conditioned by a lymphodepleting regimen in the form of cyclophosphamide and fludarabine prior to T cell infusion. Lymphodepletion probably enhances survival of the infused T cells by creating a boost in cytokines important for survival and proliferation. Lymphodepletion may also mediate a reduction of Tregs in the tumor area116,164,165. Improvements for this type of therapy involve better survival of infused cells and better homing to the tumor area166.

Genetically engineered T cells Genetically engineered T cells are yet another T cell therapy under investigation. T cells can be genetically modified by retroviral vectors to gain or enhance anti-tumor properties. Their specificity can be re-directed against a tumor antigen by insertion of a gene encoding a tumor-reactive TCR167 or the single-chain variable fragment of an antibody recognizing a cell surface molecule of tumor cells168. T cells have also been modified in order to increase their survival and function, for example by the insertion of genes encoding receptors with co-stimulatory domains, growth factors and chemokine receptors169. Suicide genes have been inserted to increase safety and constitutive or inducible markers allow tracking of infused T cell migra- tion and activation170.

33 Present investigation

General aim The aim of the project was to explore the possibility of using T cell immunotherapy as a novel treatment strategy for midgut carcinoid tumors. Metasta- sized midgut carcinoids represent a therapeutic challenge due to the excessive hormonal secretion often associated with the tumor and the low benefit of radio- and chemotherapy. Important aspects of the project were to identify potential tumor antigens and to investigate T cell and tumor immunology parameters in midgut carcinoid patients.

Specific aims

I Identify potential midgut carcinoid-associated antigens suitable for T cell immunotherapy based on neuroendocrine tissue restriction.

II Describe an identified novel splicing form of the vesicular monoamine transporter VMAT 1, denoted VMAT 115.

III Identify potential midgut carcinoid-derived HLA-A*0201-binding peptides and to investigate the possibility of tumor immune responses in patients.

IV Investigate the immunological status of midgut carcinoid patients and the presence of regulatory T cells in peripheral blood and tumor.

Materials and methods For detailed information regarding material and methods, the reader is referred to the materials and methods section in each paper.

34 Results and discussion

Paper I: Gene Expression in Midgut Carcinoid Tumors: Potential Targets for Immunotherapy Tumor and differentiation antigens expressed by midgut carcinoid tumors with a limited expression in normal tissues could be used for immunotherapy against the tumor, either by adoptive transfer of in vitro activated T cells or a modified DC vaccine. This could provide a novel treatment option for patients with disseminated disease to which there is currently no successful cure. Numerous tumor antigens have been discovered, several of which have been used in clinical trials for T cell therapy of tumors. CT antigens are known to be shared among several histologically different cancers. To our knowledge, expression of CT antigens has never been investigated in midgut carcinoid tumors. A number of differentiation markers for EC cells have been described but a complete survey of their tissue-restriction is lacking. General knowledge about gene expression in midgut carcinoid tumors is limited since there are no cDNA libraries available from tumor tissue and it is difficult to obtain sufficient amounts of normal EC cell material due to its dispersed nature. Potential antigens with ubiquitous expression in normal tissues might not be good immunological targets for two reasons: the immune response will not be restricted to the tumor cells and T cells recognizing them might be severly anergic or even deleted. In order to identify potential targets for T cell therapy of classical midgut carcinoid tumors a review of the literature on proposed enterochromaffin cell differentiation markers and general tumor antigens commonly found in cancers was undertaken. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) primers for a number of gene products of interest were designed and commercial cDNA from various normal tissues was purchased in order to perform a large screening of potential antigen candidates. RT-PCR analysis performed on a panel of primary and metastatic classical carcinoids showed a rather discouraging expression of CT antigens. The only CT antigens with a robust expression were genes of the GAGE family, which a majority of tumor specimens were positive for. To our surprise GAGE messenger RNA (mRNA) was also detected in normal stomach and pancreas, however no protein expression could be shown in line with its proposed CT profile.

35 Nearly all antigens described as over-expressed in cancers were detected in carcinoid tissue. However, with the exception of caudal type homeobox transcription factor 2 (CDX-2) and survivin, their ubiquitous and strong expression also in most normal tissues raised doubts about their antigenic potential. The majority of vital organs investigated were negative for expression of CDX-2 and survivin which made them remain interesting as antigen candidates. The proposed enterochromaffin cell-associated antigens amphiphysin, CGA, islet autoantigen 2 (IA-2), protein gene product 9.5 (PGP 9.5), synaptophysin, tryptophan hydroxylase 1 (TPH-1) and the vesicular monoamine transporters 1 and 2 (VMAT-1, VMAT-2) were all strongly expressed by the tumor with the exception of amphiphysin. CGA and synaptophysin are markers used in clinical histopathological diagnosis of carcinoids and their expression confirmed the origin of our tumor samples. CGA is considered a general marker of neuroendocrine cells and was not specifically restricted to the GI tract. PGP 9.5, once proposed as a neuroendocrine marker was ubiquitously expressed in many vital organs. Synaptophysin and VMAT-2 expression have been described in neurons as well as neuroendocrine cells and they did not show a particularly restricted tissue distribution either. IA-2, a major cell autoantigen in type I diabetes, TPH-1, an enzyme involved in serotonin synthesis and most importantly the monoamine transporter VMAT-1 had a comparatively restricted tissue distribution and were considered interesting targets. Protein expression was shown with immunohistochemistry for TPH-1, VMAT-1 and survivin. All tumors investigated showed extensive staining for VMAT-1, extensive to moderate staining for TPH and a majority of tumors were also positive for survivin. Protein expression of CGA, CDX-2 and IA-2 in midgut carcinoids have been shown by others. The level of protein expression is also of importance for immunogenicity since a sufficient amount of processed peptides must be presented on the tumor cell surface for the CTL effectors to recognize it. With this study a number of potential antigen candidates for T cell immunotherapy of classical carcinoids were identified, namely CGA, IA-2, TPH- 1, VMAT-1, CDX-2 and survivin. Immunological evaluations of these potential antigens are performed in paper III.

Paper II: Identification and Characterization of a Novel Splicing Variant of Vesicular Monoamine Transporter 1 During cloning of the full length coding sequence of VMAT-1 a shorter transcript was obtained in addition to the one previously described. After DNA sequencing and investigation of exon-intron boundaries we concluded

36 that the shorter sequence was an alternatively spliced form of VMAT1 lacking the second last exon. Full length VMAT-1 is produced from 16 exons and the alternatively spliced form was therefore denoted VMAT115. VMAT115 encodes a novel protein with an unknown function. The exclu- sion of exon 15 generates a C-terminal translated in a different reading frame compared to native VMAT-1 and the dileucin-like motif necessary for localization of the protein to the proper vesicles is lost. VMAT-1 is an integral protein in the membrane of large dense core vesicles (LDCV) in endocrine and neuroendorine cells. It belongs to a family of vesicular amine transporters (VAT) that mediates the transport of biogenic amines and acetylcholine into secretory vesicles for storage and regulated exocytosis. An electrochemical gradient generated by a vacuolar ATPase aids the transport of the cationic amines into the vesicles in exchange for protons. EC cells of the gut synthesize, store and release the amine serotonin and depend largely on VMAT-1 for its accumulation in LDCVs. VMAT-1 is also present in classical carcinoid tumors derived from these cells. RT-PCR analyses of the new alternatively spliced form of VMAT1 revealed expression of VMAT115 in classical carcinoid tumor as well as normal intestine. The RT-PCR analyses indicated that VMAT115 is expressed to a much lower extent compared to the native form in all investigated tissues. Quantitative real time PCR confirmed these data, interestingly the ratio increased in metastases compared to primary tumors in two patients where both primary tumor and metastases were available. No significant conclusions regarding relative expression and malignancy can however be made from 2 cases only. VMAT115 protein was detected in a protein lysate from BON cells and a N-terminal derived VMAT-1 antibody was used to immunoprecipitate a large amount of VMAT-1 protein before western blotting (WB). Detection using another N-terminal derived antibody revealed two bands within the same size range as WB of native VMAT1. The smaller-sized protein of weaker intensity was considered to be VMAT115 and the other protein of bigger size and stronger intensity was regarded as the native form. Since VMAT115 lacks the cellular localization signal for secretory vesicles, investigations of the cellular localization of this novel protein were performed. The VMAT1 and VMAT115 coding sequences were subcloned in pEGFP-C2 to produce EGFP-VMAT1 and EGFP-VMAT115 fusion proteins. Immunofluorescence microscopy revealed that VMAT1 as pre- dicted resides in vesicle-like structures co-localized with CGA. VMAT115 on the other hand co-localizes with the ER. This is despite the fact that VMAT115 lacks any identified ER retention signals. The alternatively translated C-terminal might also confer a different function of VMAT115 compared to the native form. The ability of serotonin uptake was investigated in VMAT115 transfected cells compared to VMAT1 transfected cells. Cells expressing the native form could readily

37 accumulate 3H-serotonin as opposed to VMAT115 expressing cells that could not. The inability of VMAT115 to accumulate serotonin is likely due to its alternatively translated C-terminal and improper cellular compartment targeting. With this study an alternatively spliced form of VMAT1 lacking exon 15 was characterized. This novel protein contains a differently translated C- terminal compared to the native form. Transfection experiments show that the protein is localized in the ER and is unable to accumulate serotonin. Our data pinpoint the importance of the C-terminal part of VMAT1 for cellular localization and function.

Paper III: CD8+ T cells against Multiple Tumor- associated Antigens in Peripheral Blood of Midgut Carcinoid Patients Identification of immunogenic epitopes of tumor-associated proteins is piv- otal when developing an immunotherapeutic strategy for midgut carcinoid tumors. T cells targeting tumor-associated proteins in metastatic lesions could provide a novel therapy for this group of patients wherein current treatments have limited curative success. The tumor area consists of a very heterogeneous cell population, apart from tumor cells, there are also stromal cells, endothelial cells and immune cells. Microdissection of solely malignant cells assures, after RNA isolation and cDNA synthesis, detection of tumor cell mRNA exclusively. By per- forming quantitative real time PCR the gene expression level of antigen can be estimated. High gene expression indicates possible presence of substantial amounts of protein, which is also presented together with MHC I molecules on the surface of the tumor cell. In the present study, metastases from midgut carcinoid patients were microdissected and investigated for gene expression of proposed antigens. Microdissection of midgut carcinoid tumor specimens and quantitative real time PCR revealed high gene expression of the proposed midgut carcinoid-associated proteins TPH-1, VMAT-1, CDX-2 and IA-2 in comparison to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the housekeeping gene used for normalization. Somewhat surprisingly, survivin expression was only detected in one metastatic lesion out of eight and to a low level. The neuroendocrine marker CGA had the strongest expression of all tumor associated proteins: the expression level was often a hundred-fold that of GAPDH. The immunogenicity of a protein is determined by its ability to be recognized by the immune system. Immune activation against tumor-associated proteins has been described in many types of cancer, and whether this is the

38 case also for midgut carcinoids has not been studied. Immunogenic proteins and T cell epitopes can be used for different immunotherapeutic strategies such as adoptive T cell transfer, DC vaccinations and genetically engineered T cells. Specific epitopes are also important when assessing therapy responses. Knowledge about MHC molecules and their protein structures, in combi- nation with peptide anchoring residues, have made predictions of MHC- binding motifs within a protein possible. Potential HLA-A*0201 binding epitopes of midgut carcinoid associated proteins were selected using peptide prediction algorithms available online. After verification of binding to HLA- A*0201 using a TAP-deficient cell line with no endogenous peptide presentation, selected peptides were tested for immune recognition using blood from midgut carcinoid patients and healthy controls. In vitro stimulations using these peptides assessed the possibility of generating peptide-specific T cells in healthy donors. A selection of 16 HLA-A*0201-binding peptides derived from 6 proposed antigens was tested for CD8+ T cell recognition in blood from midgut carcinoid patients. A well-defined population of at least 0.1% IFN-secreting cells within the CD3+CD8+ T cell population, in response to stimulation with peptide-pulsed target cells, was considered positive recognition. Healthy age-matched blood donors were used as controls. The results show that midgut carcinoid patients as a group have higher frequencies of tumor-reactive CD8+ T cells compared to healthy controls. The rate of positive recognition was 14.5% and 17.1% respectively, in two groups of patients divided by their level of tumor burden. The rate of recognition in healthy donors was 5.7%. The protein with highest overall recognition was CGA which induced IFN secretion in 21.7% of patient assays and 9.3% of healthy control assays. It is also possible to generate activated peptide-specific CD8+ T cells against CGA, TPH-1, VMAT-1, IA-2 and survivin by in vitro stimulations. CD8+ T cells from healthy donors were stimulated with peptide-pulsed CD40L-matured DCs. Peptide-specific CD8+ T cells pre-activated in vivo required only one stimulation to reach IFN-secreting frequencies above 1.50%. Peptide-specific CD8+ T cells with no or low in vivo pre-activation were restimulated 3 times with peptide-pulsed autologous cells and reached IFN-secreting frequencies of between 0.16-1.22%. In this study, a number of immunogenic epitopes derived from midgut carcinoid-associated antigens were identified. Midgut carcinoid patients display immune recognition of their tumors and have a higher rate of memory tumor-reactive CD8+ T cells compared to healthy blood donors. These T cells have great potential in T cell immunotherapeutic strategies against this type of malignancy.

39 Paper IV: Midgut Carcinoid Patients Display Increased Numbers of Regulatory T cells in Peripheral Blood with Infiltration into Tumor Tissue The general immunological balance in patients with malignancies tends to be shifted away from the beneficial cytotoxic TH1 response needed to clear transformed cells. The tumor and its microenvironment mediate strong mechanisms for immune evasion and TH1-immunosuppression. T cell immunotherapy aims at promoting TH1 responses and enhances anti-tumor CTLs. Before using patient T cells in any form of T cell immunotherapy a number of immunological parameters were examined in midgut carcinoid patients’ blood and tumor tissue. Patient blood was examined for presence and quality of Tregs. CD25 has been used as a broad marker for Tregs although present on activated effector TH cells as well. Tregs have also been described to down-regulate the IL-7 receptor alpha chain (CD127-). The intracellular antigen Foxp3 has so far been regarded as the most specific Treg marker. We found that midgut carcinoid patients display significantly higher frequencies of Tregs in peripheral blood compared to healthy blood donors based on the CD4+CD25+ and CD4+Foxp3+ phenotypes. Patients carrying a high tumor burden also have significantly higher Treg frequencies compared to low tumor burden patients. Tumor burden thus seems to influence sys- + + - temic TH1 immunosuppression. The CD4 CD25 CD127 cell population was also significantly increased in patient blood and simultaneous staining for Foxp3+ revealed that the majority of Foxp3+ cells reside within this population. A population rich in Tregs defined solely by surface markers is interesting for sorting purposes that would exclude Tregs from activated effector TH cells. T cells from patients with a high tumor burden were further analyzed regarding function. In comparison to healthy controls, patient T cells were found to be less responsive when subjected to polyclonal activation in the form of agonistic TCR antibody together with IL-2. Proliferation was measured using AlamarBlue assays and patient T cells displayed decreased proliferation also when the CD25+ fraction was separated from the CD25- fraction. CD25+ cells exhibited practically no proliferation and the CD25- cells did not demonstrate improved proliferation in the absence of CD25+ cells suggesting a sustained anergized phenotype. Regardless of any proposed marker, a Treg is defined by its ability to suppress T cell proliferation nonspecifically. Suppressive properties of patient CD25+ cells were tested in a functional assay. Patient lymphocytes were separated into a CD25+ and a CD25- fraction and added to carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled, stimulated allogeneic responder cells and proliferation was detected by flow cytometric analysis.

40 Responder cells proliferated vigorously when mixed with CD25- cells as opposed to cells mixed with CD25+ cells. Allogeneic proliferation was efficiently inhibited by patient-derived CD25+ cells down to a ratio of 10:1 and the latter can therefore be regarded as truly regulatory. Patients that exhibit higher frequencies CD25+ cells most likely have a greater proportion of Tregs in blood as well as higher Treg-mediated immunosuppression. A panel of cytokines was measured in patient sera and compared to healthy donors in order to evalute the cytokine milieu. The pro-inflammatory cytokines IL-12p70 and IL-1 were significantly lower in patients compared to controls and there was a tendency towards high TGF in patients, however not to a statistically significant level. Shifts in TH1 and TR1/ TH3 balance might be more prominent in the tumor microenvironment and less de- tectable systemically. T cells infiltrating the tumor area are interesting in many aspects. TILs have been used in adoptive T cell therapies after isolation and in vitro expansion. Presence of Tregs in the tumor area also predicts a poorer prognosis. T cell infiltration in midgut carcinoid tumor tissue was investigated by immunohistochemistry and immunofluorescence using the T cell markers CD4+, CD8+ and Foxp3+. Evaluation was performed based on presence of single, scattered cells and reactive foci infiltrating the immediate tumor area. A majority of tumors were positive for T cell infiltration with a general tendency towards heavier infiltration in metastases. CD4+-infiltrated tumors also displayed presence of Foxp3+ cells. Midgut carcinoid patients, particularly those with a high tumor burden, exhibit T cells which appear to be suboptimal for tumor clearance. Tregs and deficient effector T cells are important aspects to bear in mind when attempt- ing a T cell immunotherapy approach.

41 Conclusions

Paper I Midgut carcinoid tumors express several tissue-restricted proteins that may serve as tumor antigens in T cell immunotherapy for this type of tumor. CGA, IA-2, TPH-1, VMAT-1 and CDX-2 represent fairly restricted candidates with a strong tumor cell expression both at the mRNA and protein level. Survivin might be less beneficial due to heterogeneity in tumor expression.

Paper II The novel alternatively spliced form of VMAT1, VMAT115, lacks an exon compared to the native form and encodes a differently translated C-terminal. VMAT115 is localized in the ER and is unable to accumulate serotonin. The cellular localization and the function of VMAT115 need to be fully elucidated before determining its potential use in diagnostic, prognostic or treatment strategies.

Paper III CD8+ T cell responses in the form of IFN production were detected against HLA-A*0201-binding peptides derived from proposed midgut carcinoid antigens in peripheral blood of patients. T cell responses were detected to a greater extent in patients compared to healthy controls. Tumor-directed T cells were detected in peripheral blood of healthy controls after in vitro stimulations using the peptides. These antigens have great potential in modified DC vaccination or adoptive T cell therapy.

Paper IV Midgut carcinoid tumors are frequently infiltrated by T cells and TILs might constitute a source of anti-tumor CTLs. The tumor area is, however, infiltrated by Tregs as well and patients exhibit a systemic increase in circulating Tregs. Patient PBMCs display a decreased proliferative capacity compared to healthy donors. Tregs have an important role in suppressing anti-tumor immunity and successful T cell immunotherapy may require Treg reduction.

42 Acknowledgements

Arbetet med denna avhandling genomfördes vid enheten för klinisk immunologi, institutionen för onkologi, radiologi och klinisk immunologi, medicinska fakulteten, Uppsala universitet.

Projektet bistods av Dr. Raymond and Beverly Sackler och finansierades med anslag från the Verto Institute, Stamford, CT.

Många personer har på ett eller annat sätt bidragit med inspiration, stöd och uppmuntran under arbetet med denna avhandling och ni förtjänar alla att nämnas med stor värme och tacksamhet.

Till följande vill jag rikta ett särskilt stort tack:

Prof. Thomas Tötterman, min huvudhandledare, för att du genom ditt stöd och din uppmuntran har inspirerat mig att envist fortsätta mot doktorshatten. Tack för att du är en god lyssnare, att du ser möjligheter istället för problem, samt inte minst för att du håller GIG-gruppen och hela Klinimm på en stadig kurs framåt!

Valeria Giandomenico, min biträdande handledare som delat Carcinoid- projektets vedermödor med mig under dessa år! Tack för dina tappra försök att intressera mig för molekylärbiologi, för att du tar dig tid att minutiöst dubbelkolla alla mina -s och din fantastiska tiramisu. Stort lycka till med Carcinoid-projektet i framtiden!

Magnus Essand, vars dörr alltid står öppen för frågor och goda råd. Tack för ditt stora engagemang i mitt projekt, för att du alltid tar dig tid att disku- tera mina experiment och manuskript. Ditt intresse för vetenskap är inspire- rande och dina synpunkter har alltid varit otroligt värdefulla för mig!

Prof. Kjell Öberg, vid enheten för onkologisk endokrinologi, inst. för medicinska vetenskaper som initierade Carcinoid-projektet tillsammans med Klinimm, Monica Hurtig och Lena Olsson, fantastiska forsknings- sjuksköterskor på kliniken för onkologisk endokrinologi, Akademiska sjukhuset som bildar en ovärderlig länk mellan patienter och forskning, Prof. Emer. Lars Grimelius, vid inst. för genetik och patologi, som mer

43 än gärna delar med sig av sina otroliga kunskaper i neuroendokrinologi. Tack även till Eva Tiensuu-Jansson, Janet Cunningham, medförfattare samt Jan Saras, Åsa Forsberg och Margareta Halin-Lejonklou.

Angelica Loskog, min allra första handledare på klinimm med ett brinnande intresse för immunologi! Tack för alla idéer, inspiration och goda råd du bidragit med till mitt projekt, för alla peptalks och trevliga luncher, fika- och godisstunder. Lycka till med T-Missiler och docentur!

Stort tack till alla disputerade GIGare som banat väg före mig; Björn Carls- son, som delar mitt intresse för T celler och som frikostigt delat med sig av sina förråd av cytokiner och antikroppar, Wing-Shing Cheng, hängiven schlagerfantast som introducerade mig i viruslabbet, Christina Ninalga, som visat mig att det går att vara mamma och disputera och som vet betydel- sen av att kämpa för att nå sina mål, Helena Dzojic, en äkta Star vars leende och oförtrutet positiva inställning hållit mitt mod uppe ända sedan vi började på klinimm tillsammans, och Fredrik Carlsson, äntligen är du en av oss!

Övriga GIGare som snart vuxit i sina labrockar; Angelika Danielsson, Ole Forsberg, Arian Sadeghi, samt SatellitGIGarna Moa Fransson, Sara Mangsbo, Camilla Lindqvist och Justyna Leja som alla bidrar till den härliga stämningen i vår grupp! Tack för alla trevliga stunder vid FACSen, i cellgrottan, alla luncher o fikastunder. Lycka till med egna avhandlingar!

Berith Nilsson, för alla trevliga pratstunder i viruslabbet och särskilt tack för all din hjälp och allt du lärt mig om monoklonaler, Gabriella Paul- Wetterberg, för trivsamt sällskap i skrivrummet.

Klinimm studenter jag aldrig glömmer; Kinga Star Ziobro, Roberta Sommaggio, som flitigt arbetade med mitt projekt under min mammaledig- het, examensarbetare Rebecka Eriksson och Lisa H. Ekbom, samt Linda Gustavsson.

AnnaKarin Lidehäll och Susanne Lindblom, som kämpar tappert för att hålla Caliburn vid liv.

Ö-gruppen, särskilt tack till Ulrika Johansson för alla goda råd (och rea- gens) vad gäller immunhistokemi och hennes deltagande i min något kortva- riga ryttarkarriär på Akademistallet, tack också till Margareta Bumsan Engkvist, Peetra Magnusson, Magnus Ståhle, Annika Moëll, Andrew Friberg, Sanja Cabric, Christian Molnar, Oskar Skog, samt utflugne Peter Schmidt. Tack också till Masafumi och Megumi Goto.

Klinimm på andra sidan (C5-huset), tack till Johan Rönnelid för goda råd och deltagande vid mitt lic seminarium, tack också till Linda Mathsson,

44 Jonas Andersson, Jennie Bäck och Jenny Tjernberg samt utflyttade He- lena Johansson och Lisa Moberg

Stort tack till Mats Bengtsson och Klinimm rutin, för hjälp med HLA- typningar, samt till Maj-Britt Nordin och Maj-Britt Renlund för tålmodig hjälp med Cantos alla små egenheter.

Jan Grawé på Rudbeck Cell Analysis Core Facility, som med sina otroliga kunskaper inom flödescytometri och konfokalmikroskopi underlättat mitt arbete. Tack även till övriga medförfattare, Lars Gedda och Carina Hell- berg, för deras bidrag till denna avhandling och till Mohammad Alimo- hammadi för utbyte av peptider.

Mona Persson, Ann-Sofie Lindberg och Elin Ekberg, för att ni tar så väl hand om all administration på klinimm. Ni är guld värda! Tack även till Rudbecks IT avdelning med Viktor Persson och Per-Ivan Wyöni.

Stort tack till Blodcentralen, Akademiska sjukhuset och Karin Tapper, som försett mig med otaliga buffy coats genom åren, samt till alla friska blodgivare som generöst deltagit i min forskning.

Tack till Forskarskolan (UGSBR), Catharina Svensson och Birgitta Jön- zen, jag vill önska hela Class of 2001 ett jättestort lycka till i framtiden! Tack också till mina gamla kursare på Biomedicinarprogrammet samt nyfunna vänner på Läkarprogrammet.

Mina barndomsvänner Lena Forsmark, Jennie Zorn och Camilla Isberg som jag tyvärr inte hinner träffa så ofta som jag skulle vilja men desto roli- gare när vi väl gör det!

Mamma Karin och pappa Hans, som önskar att de hann se mer av sin dot- ter än de gör, tack för att Ni alltid ställt upp i vått o torrt, lillasyster Elin, för barnvaktning, korrekturläsning, ständig datasupport och godis, mormor Hilda för god mat och morfar Allan som ser mig bli klar från ovan samt övrig släkt o vänner hemma i Luleå.

Manuel, som tålmodigt följt med mig under hela min doktorandresa, som gjort ovärderliga inhopp vid mikroskopet, som firat vid framgång och som fällt upp ett paraply under regniga dagar och som lärt mig att man aldrig behöver ge upp, bara hitta en lättare väg. Jag hade aldrig klarat det utan dig! Oscar, som föddes lagom till halvtid, och som lärt mig att livet också består av nior, jiaffer och att hoppa i vattenpölar. Mammas lilla monster, nu ska vi ha en lång semester tillsammans! Puss till er båda!

45 References

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Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 306

Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine”.)

ACTA UNIVERSITATIS UPSALIENSIS Distribution: publications.uu.se UPPSALA urn:nbn:se:uu:diva-8421 2008