Evaluation of the Microenvironment and Immune Function in Histiocytic Sarcoma, a Tumour of Dendritic Cells

Evaluation of the microenvironment and immune function in histiocytic sarcoma, a tumour of dendritic cells

Chiara Talamonti

Downing College

Supervisors: Dr Barbara Blacklaws Dr Jane Dobson

This dissertation is submitted for the degree of Master of Philosophy Department of Veterinary Medicine University of Cambridge

March 2018

( 16,570 words)

Abstract

Canine histiocytic sarcoma (HS) is a highly aggressive tumour of histiocytic origin and it accounts for up to 50% of malignant tumours in flatcoated retrievers (FCR). Effective treatment has yet to be found, and prognosis is always poor.

In recent years, in human and veterinary oncology, there has been a growing interest in regulatory T cells (Treg). This population of cells has the ability to down-regulate the immune system, aiding tumour growth and diffusion. In the literature evidence can be found of Treg infiltrating tumours and of elevation of percentages of Treg in peripheral blood of dogs and people with cancer.

The aims of this project were to evaluate the presence of Treg infiltrating canine HS and to interrogate the tumour microenvironment to assess the molecules involved in tumour immune- evasion. Peripheral blood was analysed to determine the percentage of Treg in dogs bearing HS and age-matched controls.

In this study 27 archive samples of HS were immunolabelled with MHC class II, E-cadherin, IL-10, CD3, FoxP3 and PD-L1 (26 samples). Eight blood samples from dogs bearing HS were analysed via flow cytometry to identify percentages of T cell (CD3+CD4+ and CD3+CD8+) and + + + of Treg (CD4 , CD25 , FoxP3 ).

Findings showed no significant difference in the percentage of Treg in peripheral blood of disease FCR. However, a trend was found in a decrease of CD3+ cells in dogs with HS, driven by a reduction of CD3+CD8+ cells. Within the affected group a higher proportion of CD3+CD4- CD8- cells (possibly γδ T cells) was also observed.

Immunohistochemistry identified strong staining for MHC class II in all samples, confirming the presence of antigen presenting cells; and no staining for E-cadherin suggested mature dendritic cells as the cells of origin. Infiltrating cells stained strongly for CD3 and between 1% and 20% of these cells expressed FoxP3, confirming the presence of Treg. Surprisingly, low staining for IL-10 suggests that the release of this cytokine by Treg plays a minor role in immune evasion in canine HS.

Antibody validation for a cross-reactive anti-PD-L1 antibody was achieved through immunofluorescence analysis of frozen HS samples and assessment of mRNA expression though PD-1 and PD-L1 assays on RT-qPCR. On FFPE samples positivity for PD-L1 was found in all samples, and overall 41% to 60% of neoplastic cells expressed the marker.

This project has confirmed infiltration of Treg within the microenvironment of canine HS and has identified a possible role for the PD-1/PD-L1 pathway in canine HS immune evasion.

Preface

The study work described in this dissertation was performed at the Department of Veterinary Medicine, University of Cambridge, between April 2017 and March 2018 under the supervision of Dr Barbara Blacklaws and Dr Jane Dobson.

This dissertation is the result of my own work and includes nothing which is the outcome of the work done in collaboration except when specifically indicated in the text.

The dissertation does not exceed the word limit (20,000) for the respective Degree Committee.

The contents have not previously been submitted for any degree or any other qualification at the University of Cambridge or any other institution.

Chiara Talamonti

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Acknowledgments

Firstly, I would like to express my sincere gratitude to my supervisors Dr Barbara Blacklaws and Dr Jane Dobson for their continuous support during my MPhil project. For their patience, encouragement and incredible knowledge. I could have not asked for better supervisors.

I would also like to gratefully acknowledge the people and organisations for their help and support, without which this project would not have been possible:

The clinical pathology team at the Department of Veterinary Medicine for their great advice and suggestions, and for allowing me to spend as much time as I needed in their laboratory, and always providing delicious cake.

The pathology team at the Department of Veterinary Medicine, in particular Dr Costantino- Casas for the advice on staining protocols and grading systems and Dr Kate Hughes for the technical and moral support.

Dr Jack Robertson for all the help with immunofluorescence, immense patience and great triathlon tips.

Pet savers for funding this MPhil project and the Flatcoated Retriever Society, Liz Branscombe in particular, for all the help in collecting cases and the passionate and engaged owners.

University of Cambridge, particularly Downing College and the Department of Veterinary Medicine for the great opportunity and incredible experience.

Last but not least, I would like to thank my husband Michele, for always being by my side.

Contents

Abstract……………………………………………………………………………………i

Preface……………………………………………………………………………………iii

Acknowledgements………..……………………………………………………………..iv

List of figures………...………………………………………………………………....viii

List of tables…………………...…………………………………………………...….....ix

Abbreviations……………………………………..…………………………………...….x

Chapter 1: Introduction

1. Canine Histiocytic Sarcoma……………………………………………………………1

1.1 Myeloid cell lineage……………………………………………………………...2

1.2 Canine Cutaneous Histiocytoma…………………………………………………7

1.3 Histiocytic Sarcoma Complex……………………………………………………9

1.4 Haemophagocytic histiocytic sarcoma………………………………………….13

1.5 Predisposition in flatcoated retrievers…………………………………………..14

2. Regulatory T cells and immune tolerance…………………………………………….16

3. Regulatory T cell populations in cancer………………………………………………18

4. Aims of study………………………………………………………………………….20

Chapter 2: Materials and Methods

1. Case selection…………………………………………………………………………22

2. Sample collection……………………………………………………………………..23

3. Histopathology………………………………………………………………………...23

3.1 Preparation and staining………………………………………………………...23

4. Immunohistochemistry……………………..…………………………………………24

4.1 Antibody validation……………………………………………………………..24

4.2 Antigen Retrieval……………………………………………………………….25

4.3 Staining…………………………...…………………………………………….25

4.4 Scoring………………………………………………………………………….25

4.5 Imaging…………………………………………………………………………26

5. Immunofluorescence………………………………………………………………….26

5.1 Sample preparation……………………………………………………………...26

5.2 Antibody validation……………………………………………………………..26

5.3 Staining…………………………………………………...…………………….27

5.4 Imaging…………………………………………………………...…………….27

6. Flow Cytometry……………………………………………………………………….27

6.1 Antibodies………………………………………………………………...…….27

6.2 Sample preparation……………………………………………………………...29

6.3 Flow cytometry analysis………………………………………………………..29

7. Reverse transcription PCR…………………………………………………………….32

7.1 Sample preparation……………………………………………………………...32

7.2 Purification of total RNA from FFPE tissue……………………………………32

7.3 Primer selection………………………………………………………………....32

7.4 Reverse transcription of RNA…………………………………………………..33

7.5 RT-PCR conditions……………………………………………………………..34

8. Submarine Agarose Gels……………………………………………………………...35

9. DNA purification……………………………………………………………………...35

10. Sequencing…………………………………………………………………………35

11. Statistics……………………………………………………………………………36

Chapter 3: Results

1. Histopathology……………………………………………..………………………….37

2. Immunohistochemistry………………………….…………………………………….39

3. Flow Cytometry……………………………………………………………………….45

4. PD-L1 antibody validation…………………………………...……………………….49

4.1 Immunofluorescence…………………………………..….…………………….49

4.2 Reverse transcription PCR…………………………………………..………….51

4.3 Immunohistochemistry for PD-L1……………………………………..……….59

Chapter 4: Discussion

1. Discussion……………………………………………………………………….…….60

2. Conclusions and future work………………………………………………………….70

References………………………...……………………………………………………..72

Appendices………...…………………………………………………………………….86

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List of figures

Figure 1.1 Classification of histiocytic disease……………………………………………2

Figure 1.2 Dendritic cell lineages………………………………………………………….6

Figure 1.3 Macroscopic appearance of canine cutaneous histiocytoma on pinna…………7

Figure 1.4 Cytological evaluation of a fine needle aspirate of histiocytic sarcoma………11

Figure 1.5 Joint lesion in a flatcoated retriever…………………………………………...15

Figure 2.1 Haematoxylin and eosin staining of a histiocytic-type HS……………………22

Figure 2.2 T cell flow cytometry profile……………………………………………….....30

Figure 2.3 Treg flow cytometry profile………………………………………………...…31

Figure 3.1 Hematoxylin and eosin staining of tumour 21………………………...………38

Figure 3.2 Immunohistochemical staining for MHC class II and E-cadherin..……...……41

Figure 3.3 Immunohistochemical staining for CD3 and FoxP3..……………………...….42

Figure 3.4 Immunohistochemical staining for IL-10…………………………………...... 43

Figure 3.5 Peripheral blood lymphocyte surface marker expression……………………...47

Figure 3.6 Peripheral blood Treg marker expression……………………………………...48

Figure 3.7 Immunofluorescent staining for PD-L1……………………………..………....50

Figure 3.8 Melt curves for PD-L1 and PD-1 RT-qPCR assays………………………....…54

Figure 3.9 Melt curves for GUSB and RPL8 RT-qPCR assays………………………...... 55

Figure 3.10 Melt curves for RPL13A and RPS5 RT-qPCR assays……………………….56

Figure 3.11 Melt curves for RPS9 and HPRT1 RT-qPCR assays………………………...57

Figure 3.12 PD-1, PD-L1 and reference gene RT-qPCR products on agarose gels………58

Figure 3.13 PD-L1 staining on FFPE tissue…………………………………………...….59

viii

List of tables

Table 2.1 Summary of antibodies used for immunohistochemistry…………….………24 Table 2.2 Antibodies tested for reactivity to canine PD-L1 by immunofluorescence…..26 Table 2.3 Summary of antibodies used for flow cytometry……………………………..28 Table 2.4 Primers used for RT-PCR……………………………………………………..33 Table 2.5 Cycling conditions…………………………………………………………….34 Table 3.1 Individual scoring for each tumour with the markers shown…………………44 Table 3.2 Summary of results from T cell surface markers in peripheral blood…….…..46 Table 3.3 Summary of results from Treg markers in peripheral blood……………….…46 Table 3.4 Cycle threshold (Ct) values for assays performed..…………………………...53

Abbreviations

HS histiocytic sarcoma DC dendritic cells CCH canine cutaneous histiocytoma FCR flatcoated retriever CD cluster of differentiation GM-CSF macrophage colony stimulating-factor TNF tumour necrosis factor IL interleukin CLA cutaneous lymphocyte-associated antigen

Treg regulatory T cells FoxP3 forkhead box P3 MHC major histocompatibility complex IFN-γ interferon gamma NK natural killer cells NFAT nuclear factor of activated T cells RUNX-1 runt-related transcription factor-1 iTreg induced regulatory T cells PD-1 programmed cell death protein 1 PD-L1 programmed cell death protein ligand 1 COX-2 cyclooxygenase-2 PGE-2 prostaglandin E2 TGFβ transforming growth factor beta IDO indoleamine 2, 3-dioxygenase FFPE formalin-fixed paraffin embedded H&E haematoxylin and eosin IgG immunoglobulin G HRP horseradish peroxidase

PFA paraformaldehyde PBS phosphate-buffered saline NTC no-template control Ta annealing temperature Bp base pair ND not detected

Chapter 1: Introduction

1. Canine Histiocytic Sarcoma

Canine histiocytic sarcoma (HS) is a highly aggressive tumour of histiocytic cell origin. It is thought to arise from cells of the myeloid lineage, including macrophages and dendritic cells (DC) (Hafeman et al., 2010; Ito et al., 2013; Moore et al., 2006). It belongs to the class of canine histiocytic disorders, a very heterogeneous group of diseases that differ from one another in clinical behaviour and pathological features (Abadie et al., 2009; Affolter and Moore, 2002; Moore, 2014) (Fig 1.1). Also included in this class are canine cutaneous histiocytoma (CCH) and reactive histiocytosis. The first, a benign neoplasm of the skin which usually spontaneously regresses (Affolter and Moore, 2002; Moore, 2014). The latter is thought to arise from an immune system dysregulation and is described in two distinct forms, cutaneous and systemic (Affolter and Moore, 2002).

Whereas the benign neoplastic form, CCH, has a high incidence rate (standardised incidence rate of 337 per 100,000 dog/year) (Dobson, 2013), HS is relatively infrequent within the canine population. However, there is a high prevalence in certain breeds (Kennedy et al., 2016). The two over-represented breeds are the Bernese mountain dog and the flatcoated retriever (FCR) (frequency of 25% and 20% respectively) (Abadie et al., 2009; Affolter and Moore, 2002; Dobson et al., 2006). Other breeds commonly affected are the rottweiler, golden retriever, Labrador retriever (Affolter and Moore, 2002; Moore, 2014), and, less so, the Pembroke Welsh corgi and schnauzer (Ito et al., 2013).

There are diverse clinical presentations and biological behaviour amongst canine HS, this has generated the term “histiocytic sarcoma complex” (Fulmer and Mauldin, 2007; Moore, 2014). The definition of histiocytic sarcoma complex has evolved in time, and the exact classification is still not defined. Two forms have been recognised, a localized and a disseminated one, however it is unclear if these

Chapter 1: Introduction

are two distinct presentations. The current belief is that the two forms represent an early and late stage of the same pathological process (Boerkamp et al., 2013; Kennedy et al., 2016). They present identical histopathological features but exhibit variable clinical behaviour (Affolter and Moore, 2002).

NON MALIGNANT MALIGNANT NON NEOPLASTIC NEOPLASTIC • Reactive histiocytosis — Localized histiocytic — Cutaneous sarcoma histiocytosis — Disseminated — Systemic histiocytic sarcoma histiocytosis

NEOPLASTIC — Cutaneous histiocytoma

Figure 1.1: Classification of histiocytic disease, adapted from “Canine Histiocytic Disease” (Coomer and Liptak, 2008). Canine histiocytic disease are a heterogeneous class of disorders. They can be classified as malignant and non-malignant. The latter comprehends reactive histiocytosis and cutaneous histiocytoma.

1.1 Myeloid cell lineage

Histiocytes are a bone marrow derived subgroup of leucocytes that play a pivotal role in the functioning of the immune system. They originate from a common cluster of differentiation (CD) 34+ precursor in the bone marrow that differentiates into the myeloid lineage then into macrophages and DC (Fig 1.2).

Macrophages are released into the blood stream from the bone marrow as monocytes, and reach various tissues throughout the body, where they enter and differentiate into mature macrophages. Tissue resident macrophages can be found

Chapter 1: Introduction

in all tissues with recruitment of additional macrophages from the blood during inflammation (Fulmer and Mauldin, 2007).

Macrophages are specialized in phagocytic activity. If stimulated they can also present antigens to previously activated or memory T cells. However they are less efficient in this function than DC and so cannot stimulate naïve T cells. (Affolter and Moore, 2002; Moore, 2014; Moore et al., 2006; Rossi et al., 2009).

Dendritic cells, under the influence of granulocyte-macrophage colony stimulating- factor (GM-CSF), tumour necrosis factor α (TNF-α) and interleukin (IL) 4, then differentiate into CLA+ (cutaneous lymphocyte-associated antigen) intraepithelial DC named Langerhans cells or CLA- interstitial DC (Fulmer and Mauldin, 2007; Moore, 2014).

Langerhans cells are localized in the epidermis and can be found in the epithelia of skin and the alimentary, respiratory and reproductive tracts (Merad et al., 2008; Moore, 2014). Interstitial DC are localized in the dermis, alongside post capillary venules, and in almost every tissue of the body, excluding the brain. But they can be found in the meninges and choroid plexus (D’Agostino et al., 2012; Moore, 2014). DC may also be found in peripheral lymphoid organs, such as lymph nodes and spleen. Here they are described as interdigitating DC. In the lymph nodes the population is divided into resident DC and migratory DC; the latter travel from surrounding tissue via the afferent lymphatics and belong to both the Langerhans and interstitial cell populations (Moore, 2014; Shortman and Naik, 2007).

DC belong to the family of leukocytes and are potent stimulators of adaptive immunity. They transfer information from the outside environment to the cells of the adaptive immune system (Banchereau et al., 2000). In addition, they are also capable of preventing, inhibiting or modulating T cell-mediated effector responses allowing maintenance of immunological tolerance (Steinman et al., 2003). These cells are able to carry out a broadly attenuating effect through production of

pleiotropic anti-inflammatory factors and by inducing regulatory T cells (Treg). Additonally they can present self-antigens to lymphocytes in the absence of

Chapter 1: Introduction

costimulation thereby leading to peripheral tolerance (Maldonado and Andrian, 2010).

Immature DC are poorly immunogenic, as their role is to acquire antigenic material from the tissue they are resident in. Because of this they express only modest levels of major histocompatibility complex (MHC) class II molecules. Their ability to detect pathogen infection relies on the use of specific receptors that detect pathogen associated molecular patterns, and damage associated molecular patterns, released by tissue following cellular distress (Maldonado and Andrian, 2010).

As a consequence of inflammatory stimulus DC mature, during migration from the periphery to the draining lymph node, through the lymphatics. When undergoing the complex process of maturation DC upregulate MHC expression, gaining the capacity to process antigen and stimulate T cells (Banchereau et al., 2000). In immature DC most MHC molecules are stored intracellularly within vesicles. With maturation these are then transported to the cytoplasmic membrane where MHC molecules are found in mature DC (Pires et al., 2013). The upregulation of chemokine receptor CCR7 allows the DC to migrate to the draining lymph node where they present the antigenic cargo to T cells thus activating them (Alvarez et al., 2008). In addition to the immunogenic activity, these cells are capable of

promoting immunotolerance by either converting naïve T cells to assume a Treg

phenotype or by promoting Treg differentiation (Maldonado and Andrian, 2010). Evidence supports that both mature and immature DC can provide immunological

tolerance through Treg (Lambrecht and Hammad, 2009).

Because of the morphological similarities between different histiocytic cells, immunophenotyping of surface markers has been used over the years to aid in the distinction of the various cells. However, macrophages and DC have very plastic marker expression profiles, often changing the molecules they express in response to different stimuli. This can make it very difficult to definitively identify different cell types and often multiple markers are used to classify the cells (Fulmer and Mauldin, 2007; Kennedy et al., 2016) .

Chapter 1: Introduction

The molecules responsible for antigen presentation to T cells are MHC class I and II and CD 1 (Looringh Van Beeck et al., 2008; Moore et al., 2017; Moore, 2014). Hence, DC can be identified as a result of their high levels of expression of these molecules. MHC class I and II are expressed on DC in all tissues and are upregulated in response to inflammatory stimuli. CD1 proteins are mainly expressed by DC of the skin. DC also express co-receptors for antigen presentation (necessary for T cell activation via CD28) such as CD80 and CD86 as well as other stimulatory or inhibitory molecules (Looringh Van Beeck et al., 2008; Moore, 2014). As a result of the different role played in the immune response, macrophages express slightly different markers to DC. These are low and inconsistent levels of MHC molecules, CD1 and CD11c and high expression of CD11b, or CD11d in hematopoietic sites (Looringh Van Beeck et al., 2008; Moore, 2014; Strominger, 2010).

Identification of Langherans cells may be achieved through their expression of E- cadherin, a calcium-dependent adhesion molecule. E-cadherin is important in adhesion between cells of the epithelium, but its cytoplasmic domain also interacts with beta catenin which is important in both cell-cell adhesion and as a signal transducer for the Wnt signalling pathway (Moore et al., 1996).

In the dog, Thy-1 is expressed by dermal DC, aiding distinction from epidermal Langherans cells (Moore, 2014). Thy-1 (CD90) is a heavily glycosylated glycophosphatidylinositol anchored cell surface protein expressed by myeloid cells, among other cells. Because Thy-1 functions as a mediator in the adhesion of mononuclear cells to endothelial cells and fibroblasts, it is abundant in cells in connective tissues and post-capillary endothelium (Schubert et al., 2011).

Other transmembrane receptors are also engaged in the characterization of cells of myeloid origin. The receptors of the beta-integrin family aid adhesion between cells and also between cells and the extra-cellular matrix (Moore, 2014). They are heterodimers made up of an alpha and beta subunit. There are different types of beta subunit and these can pair with many different alpha subunits. CD18 is the beta2-integrin subunit and is expressed by all leukocytes (Mazzone and Ricevuti, 1995). Differentiation of cell type is possible using this marker because

Chapter 1: Introduction

macrophages and DC have a much stronger expression of CD18 in comparison with other leukocytes, allowing classification. Furthermore, macrophages have more marked expression of CD18 compared to DC. Consequently, high levels of expression of CD18 with low levels of MHC class II identifies a macrophage (Moore, 2014).

The α subunits (CD11 subtypes) that complex with beta2-integrin are also utilized to differentiate haemopoietic cells. Macrophages usually express CD11b, and CD11d when they are in the splenic pulp. CD11c is instead expressed by DC (Moore et al., 2006).

Figure 1.2: Dendritic cell lineages from “Immunesurveillance by dendritic cells: potential implication for immunotherapy of endocrine cancers” (Schott, 2006).

Chapter 1: Introduction

1.2 Canine Cutaneous Histiocytoma

Canine cutaneous histiocytoma is a benign epidermal neoplasm which arises from dermal precursors of Langerhans cells and then invades the epidermis (Fulmer and Mauldin, 2007; Moore, 2014; Pires et al., 2013). This benign neoplasm is usually recognised in patients younger than three years-old, although it has also been reported in older dogs (Fulmer and Mauldin, 2007; Moore, 2014).

CCH is prevalent in brachycephalic breeds such as boxers and bulldogs, although dachshunds, Scottish terriers, doberman pinchers and cocker spaniels are also overrepresented (Fulmer and Mauldin, 2007; Moore, 2014; Taylor et al., 1969). The tumour presents as a solitary, painless and non-pruritic epidermal lesion (Fig 1.3). The most common location is the head or the pinna, and it is also often described on limbs, but it can arise anywhere on the body (Fulmer and Mauldin, 2007) .

A B

Figure 1.3: A, B Macroscopic appearance of CCH on pinna. Courtesy of Dr D Grant.

Diagnosis can be usually achieved via cytology, but immunohistochemistry is needed for confirmation. Cells characterizing these tumours are described as round or slightly oval, with round to oval nuclei that are occasionally indented and eccentric. The nuclear:cytoplasmic ratio may vary, but is usually just above 1:1 (Fulmer and Mauldin, 2007).

Chapter 1: Introduction

Histologically, histiocytes appear arranged in uniform sheets that penetrate the dermis or subcutis. Immunohistochemical analysis demonstrates the presence of leucocyte antigens such as MHC class II, CD1, CD11c and E-cadherin. E-cadherin expression is unique to Langherans cells, although in CCH the staining pattern is not uniform and may be limited to the area adjacent to the epidermis (Fulmer and Mauldin, 2007).

Treatment is usually not necessary as the lesion initially undergoes rapid growth, reaching a diameter that ranges from 0.2 to 3 cm, and then spontaneously regresses within a few months (Fulmer and Mauldin, 2007; Pires et al., 2013; Taylor et al., 1969). In less than 1 per cent of cases multiple masses have been described, shar- peis are reported to be predisposed to this presentation. Multiple lesions may persist for longer and the regional lymph nodes tend to be involved (Moore, 2014). Treatment is considered in older dogs diagnosed with canine cutaneous histiocytoma, especially if the lesion does not regress within 2 to 3 months. Surgery and cryosurgery are curative with an excellent outcome (Fulmer and Mauldin, 2007).

The fascinating regression pattern of this tumour has interested many researchers, but is yet not fully understood (Kaim et al., 2006). A recent study found that tumour cells of CCH are immature in early lesions and then undergo maturation, expressing MHC II on the cellular membrane (Pires et al., 2013). The authors suggest that the migration of MHC vesicles to the cell periphery seems to be a significant factor in initiating regression. The initial response appeared to involve CD4+ T cells. These helper cells are capable of activating tumour specific lymphocytes and recruiting non-specific effectors such as macrophages, which exert additional tumouricidal activity by synthesis of reactive metabolites. The up-regulation of iNOS, an enzyme that produces the cytotoxic metabolite nitric oxide, also suggests that macrophages are involved in immune response and tumour regression (Kaim et al., 2006). Following the initiation of the response by CD4+ T cells, the lymphocytes mainly involved are CD8+ T cells; these are cytotoxic cells capable of mediating regression (Moore, 2014). During regression, the immune response is observable histologically as an accumulation of mature lymphocytes at the periphery and subsequently within the tumour (Kaim et al., 2006)

Chapter 1: Introduction

Therefore, this DC tumour appears to have the unique capacity to prime T cells leading to its own eradication (Kaim et al., 2006), although resident DC might be responsible for the immune response (Moore, 2014).

Different molecules have proven to be involved in the regression of CCH. A study published in 2006 analysed frozen tumour tissue to detect cytokine mRNA by semiquantitative RT-PCR (Kaim et al., 2006). During regression of the tumour an increased expression of IL-2, TNF-α and interferon gamma (IFN-γ) was identified. It is known in other systems that IFN-γ intensifies the cytotoxic potential of CD8+ T cells. Moreover the up-regulation of TNF-α promotes proliferation of T cells and natural killer cells (NK). Independently TNF-α is capable of inducing apoptosis. It is hypothesised that IL-2 aids the regression process by generating NK cells. However, what sets the regression in motion remains unknown.

1.3 Histiocytic Sarcoma Complex

Histiocytic sarcoma arises from interstitial DC. The presentation of the tumour is described as localized when it originates at a single tissue site or in a single organ. (Affolter and Moore, 2002; Dervisis et al., 2016). The disseminated presentation, historically referred to as malignant histiocytosis, (Moore, 2014; Rosin, 1986) is described as a multifocal disease with masses occurring simultaneously at multiple sites (Kennedy et al., 2016). Because of the natural distribution of DC, lesions have been identified in all tissues. Frequent locations are spleen, liver, lymph nodes, bone marrow, lung, central nervous system, intervertebral soft tissue, skin, deep fascia of muscle, and periarticular soft tissue (Chandra and Ginn, 1999; Moore, 2014). HS has been described in many breeds, although retrievers, Bernese mountain dogs and rottweilers are overrepresented. Animals are usually 6 to 11 years-old at the time of diagnosis, and there is no sex predisposition reported (Affolter and Moore, 2002; Coomer and Liptak, 2008).

Chapter 1: Introduction

Clinical signs are usually vague. Dogs present with anorexia, lethargy and weight loss (Coomer and Liptak, 2008; Moore, 2014). A common finding in animals affected by disseminated HS is anaemia. A mild non-regenerative anaemia is encountered when the bone marrow has been infiltrated, whereas a more severe regenerative anaemia is due to erythrophagocytosis (Dobson et al., 2006). Other symptoms, such as dyspnoea, lameness and neurological signs, are a consequence of localisation of mass (Moore, 2014).

Clinical staging of the tumour is vital to devise a correct treatment plan. Abdominal ultrasonography, chest radiography and bone marrow aspiration should be performed. Additionally findings from cytology and immunohistochemistry are considered (Coomer and Liptak, 2008; Moore, 2014).

Abdominal ultrasonography allows lesions within the abdominal organs to be identified, however used alone it is non-specific. The technique is beneficial as it allows image-guided fine needle aspiration (Ramirez et al., 2005). Chest radiographs may reveal pulmonary consolidation, nodular opacities or diffuse interstitial infiltrations and pleural effusion (Coomer and Liptak, 2008; Tsai et al., 2012).

Aspiration of the tumour yields high amounts of cells, these are round or spindle- like and they contain copious amounts of basophilic cytoplasm (Fig 1.4). Nuclei are eccentric, often indented, and with prominent and sometimes multiple nucleoli (Brown et al., 1994). Anisokaryosis and anisocytosis are commonly present as are a large proportion of mitotic figures. Frequently phagocytosis of erythrocytes and neutrophils by the tumour cells is observable (Fulmer and Mauldin, 2007).

Chapter 1: Introduction

A B

Figure 1.4: A (20x), B (50x). Cytological evaluation of a fine needle aspirate of HS. Sample collected from subcutaneous tissue on thorax. Discrete round cells with abundant cytoplasm with frequent vacuoles are noticeable. Marked criteria of atypia including coarse chromatin, anisokaryosis, anisocytosis, prominent nucleoli and abundant mitotic figures. Nuclei are eccentric and occasionally indented. Courtesy of Dr C Hare.

Histologically, HS present as highly cellular masses. The histiocytes are pleomorphic and their nuclei vary in shape and size, with prominent nucleoli. Multinucleated cells are often visible; abundant mitoses and bizarre mitotic figures are observed (Affolter and Moore, 2002; Chandra and Ginn, 1999; Coomer and Liptak, 2008; Fant et al., 2004).

Diagnosis is confirmed through immunohistochemistry of histopathological samples. HS expresses CD1, CD11c, CD18, CD45 and MHC class II. However, antibodies for all these antigens are not currently available for formalin-fixed tissue. Therefore, in practice, diagnosis is based on morphological features and positivity for CD18 and MHC class II, and negativity for CD3 and CD79a. As CD18 is found on all bone marrow derived leukocytes, differentiation of histocytic neoplasms from lymphomas and other round cell tumours may sometimes be challenging. A recent study has investigated the value of the macrophage scavenger receptor (CD204) as a marker for neoplastic histiocytes in dogs. It was found that resident and infiltrating macrophages also expressed CD204, so differentiation with neoplastic cells was conducted observing morphological features (cell atypia and signs of malignancy) (Kato et al., 2013). Recently a new marker, Iba1 (ionized calcium binding adapter molecule 1) has shown promising results on formalin-fixed tissue, offering support

Chapter 1: Introduction

for diagnosis of HS (Pierezan et al., 2014). Efforts are being made to investigate molecular characterisation to aid in definite diagnosis (Kennedy et al., 2016).

Treatment for HS usually involves surgery, chemotherapy and radiation therapy. However, because of the high percentage of distant metastases (over 60%) reported in localized HS, the efficacy of surgery is doubtful. Protocols using lomustine as a single agent or together with doxorubicin are commonly recommended. Because both these drugs can be myelosuppresive, a protocol alternating the two drugs (every 2 weeks) is suggested (Cannon et al., 2015). Bisphosphonates have been studied extensively for their ability to deplete macrophages and appear to be beneficial in canine HS. A recent study reports the benefits of using clondronate added to vincristine when bony involvement is present, because of its ability to reach high concentration in bones. Zolendronate in combination with doxorubicin may be beneficial for visceral tumours as it appears to increase tumour cell permeability to doxorubicin (Hafeman et al., 2012). Response to radiotherapy is usually rapid; the draining lymph node is commonly treated together with the primary lesion (Affolter and Moore, 2002; Fidel et al., 2006; Moore et al., 2017; Moore, 2014).

Mean survival for animals with localized HS in the limbs (up to 6 months) is mildly better compared to visceral forms involving the spleen (1 month) (Affolter and Moore, 2002; Craig et al., 2002; Fulmer and Mauldin, 2007; Spangler et al., 1994). This is probably due to the clinical signs related to splenic involvement, lethargy, severe anemia and exercise intolerance. When deep muscle and joints are affected the clinical sign is usually lameness which may be managed with non-steroidal anti- inflammatories. However the clinical outcome for dogs with masses affecting the limbs is also poor because of the rapid and often painful progression of the disease (Marcinowska et al., 2017). Because of the poor prognosis often euthanasia is performed if lesions are disseminated at time of diagnosis (Fulmer and Mauldin, 2007; Marcinowska et al., 2017).

Chapter 1: Introduction

1.4 Haemophagocytic histiocytic sarcoma

Haemophagocytic HS differs from all other forms of canine histiocytic disease because of its cell of origin. In contrast with the localized and disseminated form of the histiocytic sarcoma complex, which arise from antigen presenting DC, this form originates from macrophages. In particular this tumour arises from splenic red pulp and bone marrow macrophages (Kennedy et al., 2016; Moore, 2014; Moore et al., 2006).

The affected breeds are the same as with the other presentations of histiocytic sarcoma complex. There is an over-representation of Bernese mountain dogs, retrievers, rottweilers and schnauzers. The range of age at time of diagnosis is very broad (2.5 to 13 in one study) and there is no sex predisposition (Moore et al., 2006).

Macroscopically there is no distinct mass formation, since the lesions are diffuse. The typical sites of development are bone marrow, spleen, lung and liver. Initiation appears to be parallel in bone marrow and spleen, whereas the liver is reached by neoplastic cells migrating through the portal vein. Splenomegaly and hepatomegaly are recurrent findings, multiple nodular masses are frequently found in both organs. Multiple white coloured lesions have also been described in the lung (Moore et al., 2006).

Cytological presentation of the histiocytes varies from the appearance of normal macrophages to atypical cells displaying hyperchromatic nuclei, anisocytosis and anisokaryosis. Multinucleated cells are sporadically encountered, these are limited to the bone marrow (Moore et al., 2006).

Dogs affected present with anorexia, weight loss, lethargy and pale mucous membranes. Blood work-up highlights a moderate to marked regenerative anaemia and thrombocytopenia. Hyperbilirubinaemia is also a common finding, although jaundice is rare. Frequently animals exhibit hypoalbuminaemia and hypocholesterolaemia (Moore, 2014; Moore et al., 2006).

Chapter 1: Introduction

A successful treatment protocol has yet to be found, and dogs affected have a median survival time of 4 weeks, making this the malignant histiocytic neoplastic disease with the worst prognosis (Moore et al., 2006).

1.5 Predisposition in flatcoated retrievers

Flatcoated retrievers are a small population of dogs that can be found in Europe and America. Over the past few decades a concern over the predisposition to neoplasia of the modern FCR has prompted further research on the topic (Morris et al., 2002). The breed has been shown to have a high incidence of HS, as it accounts for 36 to

50% of all malignant tumours in this breed. A study conducted in the Netherlands suggested that this tumour is responsible for up to 46% of deaths in the breed and is responsible for the two-year reduction in life-span compared to other breeds (Erich et al., 2013; Kennedy et al., 2016). Mean age of diagnosis is 8.2 years and no sex predisposition has been confirmed (Fidel et al., 2006).

The FCR is most commonly affected with the localized form of HS (Kennedy et al., 2016). The primary location of the tumour in this breed is deep in the musculature, fascia or in the soft tissue surrounding the joints. The front limbs are more frequently affected. The second most common location has been found to be the spleen, this presentation is described as visceral (Fidel et al., 2006; Marcinowska et al., 2017; Moore, 2014). Both forms are highly malignant and have a high metastatic rate (Affolter and Moore, 2002; Marcinowska et al., 2017; Mariani et al., 2015).

A study conducted within our department classified histiocytic sarcoma in FCR by histopathological presentation. The tumours are described as histiocytic subtype when over half of the neoplastic cells are histiocytic and as histiocytic-spindle- pleomorphic type when the majority of cells exhibited fibroblatic-like features (Constantino-Casas et al., 2011).

Chapter 1: Introduction

Most commonly, clinical signs are related to the localization in the skeletal muscle and joint (Fig 1.5). Dogs present with lameness and pain on manipulation, in some cases swelling of the joint is noticeable on clinical examination (Fidel et al., 2006). The dogs affected with splenic HS frequently present with a palpable abdominal mass, weight loss, lethargy and exercise intolerance. Dogs presenting with inappetence, pyrexia, vomiting, melena, pallor, collapse, regurgitation and polydipsia/polyuria have also been reported in the literature (Marcinowska et al., 2017).

Blood testing may reveal anaemia and, as previously described, this may be regenerative if associated with haemophagocytosis or non-regenerative if caused by bone marrow infiltration (Dobson et al., 2006; Fidel et al., 2006). Hypoalbuminaemia, neutropaenia, thrombocytopaenia and elevation of liver enzymes have also been described (Marcinowska et al., 2017).

Figure 1.5: Joint lesion in a FCR. Radiographically the swelling of soft tissue is evident (arrow). Courtesy of Dr J Dobson.

Chapter 1: Introduction

2. Regulatory T cells and immune tolerance

Historically described as suppressor T cells, Treg are important in controlling the

potency of the immune response. Natural Treg originate from the thymus and play a key role in avoiding misguided or excessive immune reactions (Knueppel et al., 2011; Lio and Hsieh, 2008; Maldonado and Andrian, 2010; Sakaguchi et al., 2008).

A second subset of these cells does not emerge from the thymus as Treg, but instead the T cell undergoes a conversion process peripherally after activation, when the

environment is rich in Th2 cytokines (induced Treg, iTreg). These are reportedly + - peripheral CD4 CD25 T cells that are converted to iTreg and present all the features

of natural Treg (Apostolou and von Boehmer, 2004; Chen et al., 2003; Garden et al., 2011).

The immune down-regulation performed by these cells is fundamental to avoiding autoimmune disease, and normal physiological responses escalating to pathology. + + CD4 CD25 Treg target T cells and antigen presenting cells (Beissert et al., 2006;

Belkaid and Rouse, 2005; Pinheiro et al., 2011). The activity of Treg is described as “dominant”, as they actively keep in check other T cells. There are also “recessive” mechanisms that rely on cell intrinsic control, receptor editing and increase of the activation threshold. Cell intrinsic control is based on the ability of immature lymphocytes to die by apoptosis if exposed to self-antigens. The process by which self-reactive T cells are replaced by non-reactive ones is labelled “receptor editing” (Sakaguchi et al., 2008).

Similarly to other immune cells, Treg migrate and become activated in regional lymph nodes when they come into contact with antigens. They also migrate to sites of infection, inflamed tissue and tumours (Belkaid and Rouse, 2005). Compared to

other T cells, Treg need a lower concentration of antigen to be stimulated (Sakaguchi et al., 2008).

+ Treg express their activity in multiple ways. They have the ability to kill CD8 T cells, or to suppress the toxicity of these cells through a mechanism involving TGF-

Chapter 1: Introduction

β. Furthermore Treg have the power to modulate the activity of DC through the use of cytokines such as IL-10 and IL-35, or through cell-to-cell contact (Belkaid and Rouse, 2005; Maldonado and Andrian, 2010). In the latter case, following antigenic stimulation, they surround the DC, competing with the naïve T cells, and inhibiting the antigen presenting cell’s function (Hatziioannou et al., 2017; Sakaguchi et al., 2008).

The most reliable marker to characterize Treg is the transcription factor forkhead box

P3 (FoxP3). FoxP3 is fundamental for the development and function of Treg (Fontenot et al., 2003). It is able to influence other transcription factors, such as the nuclear factor of activated T cells (NFAT) and the runt-related transcription factor 1 (RUNX1) with which it can directly interact. The aforementioned transcription factors facilitate the activation and differentiation of effector T cells. As a result of the interaction with FoxP3, the transcription of effector T cells is modified and the

cells are functionally converted to Treg (Fontenot et al., 2003; Sakaguchi et al., 2008).

Another molecule that is vital for the function of Treg is IL-2, and Treg commonly express the interleukin’s receptor alpha chain, CD25 (Sakai et al., 2018). IL-2 has

a major influence on the proliferation and differentiation of Treg and is required for sustained expression of FoxP3. The cytokine is produced by activated non-

regulatory T cells; by promoting the expansion and activation of Treg it limits the expansion of non-regulatory T cells. This creates a feed-back mechanism between the two cell populations (Sakaguchi et al., 2008).

The programmed cell death protein 1 (PD-1) is a cell surface receptor that is involved in promoting self-tolerance by limiting the activity of T cells during inflammatory responses (Freeman et al., 2000). The axis it forms with its ligand, the transmembrane protein PD-L1, is an immune checkpoint pathway that plays a critical role in the prevention of autoimmunity. It does so by promoting apoptosis in antigen specific T cells in lymph nodes, and by regulating the development,

maintenance and function of iTreg (Francisco et al., 2009; Hartley et al., 2016). PD-

1 is highly expressed on Treg, and expression is induced on activated T cells. (Duraiswamy et al., 2013). PD-L1 expression is increased on many tumours and

Chapter 1: Introduction

has been associated with immune escape (Angelo et al., 2015). Expression has also been found on tumour-infiltrating lymphocytes and tumour-associated macrophages (Hartley et al., 2016). In human medicine expression of PD-L1 has been correlated with poor prognosis in many tumours, such as colon, cervical, breast, ovarian and non-small cell lung amongst others (Hartley et al., 2016; Shi et al., 2013; Muenst et al., 2014). In a recent study expression of PD-L1 in cell lines of canine tumours was investigated. HS was found to express the highest levels under basal conditions (Hartley et al., 2016).

Treg have been known and studied in dogs since the 1980s, initially the research was focused on German shepherds with progressive myelopathy, and later on patients presenting with atopic skin disease. More recently research interest has focused on

the variations in proportions of Treg in dogs with cancer (Garden et al., 2011; Rissetto et al., 2010).

3. Regulatory T cell populations in cancer

Over the past decades studies in human oncology have focused on the role of Treg in cancer patients. Histopathological analyses have confirmed that immune cells often infiltrate the tumour microenvironment (Carvalho et al., 2014; Sakai et al.,

2018). Reports of high infiltration of Treg being associated with poor prognosis in human cancer suggested that CD4+CD25+ T cells decrease anti-tumoral activity. Although, the mechanisms by which this is achieved are not fully understood. (Liu et al., 2016; Sakaguchi et al., 2008; Sakai et al., 2018; Wang et al., 2012). The influence of these cells on the immune system, as previously described, prevents harmful and excessive immune responses, however it also interferes with the advantageous response to neoplastic growth and spread (Biller et al., 2007). As in

humans, in dogs a high number of infiltrating Treg has been observed in numerous tumours including seminoma, intestinal lymphoma, mammary carcinoma and HS (Carvalho et al., 2016; Kim et al., 2013; Maeda et al., 2016; Marcinowska et al.,

2017). Furthermore in mammary carcinoma a higher infiltration of Treg has been

Chapter 1: Introduction

associated with poor prognostic factors such as high histological grade, lymphatic invasion and tissue necrosis (Kim et al., 2012; Sakai et al., 2018).

Positivity to FoxP3 in infiltrating lymphocytes of HS of FCR has been assessed by a recent study which found a significant difference between the expression of this marker in lymphocytes in soft tissue lesions (19%) and splenic masses (1%), suggesting an influential role of the microenvironment of the tissue of origin (Marcinowska et al., 2017).

In human oncology, an increased percentage of Treg in peripheral blood has been reported in multiple neoplasms including malignancies of stomach, pancreas, liver and breast to mention some (Hatziioannou et al., 2017; Ichihara et al., 2003;

Liyanage et al., 2002). Although a direct correlation between elevated Treg in the blood and disease stage has still not been confirmed, an influence on prognosis has been described (Hatziioannou et al., 2017).

A flow cytometry study identified CD4+FoxP3+ T cells in the peripheral blood of healthy dogs. 4.3% of T cells were identified as CD4+FoxP3+, this value rose to 7.5% in dogs affected by cancer. A similar increased was shown in lymph nodes, where the normal percentage was found to be 9.8%, and the percentage in tumor- draining lymph nodes was 17.1% (Biller et al., 2007). In all tumour types the presence of metastasis was correlated with an even higher frequency of CD4+FoxP3+ cells (Garden et al., 2011).

Natural Treg are not the only suppressor T cells found in tumours. Many studies have

reported the presence of iTreg. Cells within the tumour microenvironment appear to be responsible for the conversion of FoxP3- to FoxP3+ cells. Effector molecules demonstrated to be involved in the process are cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE-2), and transforming growth factor beta (TGF-β) (Hatziioannou et al., 2017).

Indoleamine 2, 3-dioxygenase (IDO), an immunomodulatory enzyme, also appears

to enhance the functional tolerance promoted by Treg through many paths. IDO-

Chapter 1: Introduction

+ expressing DC have been shown to induce the conversion of CD4 T cells to iTreg (H. Munn, 2011).

The interest in the role of this cell population in cancer has been encouraged by the prospect of effective immunotherapy treatments. Studies in mice have shown that

reducing the activity of Treg, by decreasing the cell population, induces autoimmune disease, but it also promotes effective anti-tumour immunity (Beissert et al., 2006; Knueppel et al., 2011). The therapeutic potential is great, but a balance between

reducing the numbers and attenuating the activity of Treg in cancer patients, and mitigating the consequent autoimmunity is key (Garden et al., 2011).

Current cancer treatments have been shown to affect the presence and function of

Treg, diminishing their effect. Some examples are low doses of cyclophosphamide, an immune suppressor; fludarabine and gemcitabine, which inhibit cellular DNA synthesis; paclitaxel, a mitotic inhibitor; and tyrosine-kinase inhibitors

(Hatziioannou et al., 2017). Specific therapies targeting molecules expressed by Treg have been identified in experimental models, and clinical trials in humans are ongoing (Bulliard et al., 2014; Curti et al., 2013).

The importance of understanding the mechanisms of cancer immune evasion and

the role played by Treg is meaningful to help develop therapeutic treatments in veterinary oncology and as an animal model for human research (Garden et al., 2011).

4. Aims of study

The aim of this study was to investigate the relationship between tumour, microenvironment and immune system in canine HS. This was implemented + through assessment of the proportion of FoxP3 T cells in peripheral blood of FCR diagnosed with HS, and comparison with unaffected age-matched controls. The microenvironment of HS was investigated through immunohistochemical labelling of immune suppressive markers. Histiocytic cells and infiltrating lymphocytes were

Chapter 1: Introduction

interrogated for FoxP3, PD-L1 and IL-10 among other markers. Immunofluorescence and RT-qPCR were used to validate the immunohistochemical finding for PD-L1. RT-qPCR was also helpful in comparing expression levels of PD-1 in tumour and healthy canine tissue. A better understanding of the complex interactions occurring in HS might better inform on prognosis and lead to innovative and effective therapies in the future.

Chapter 2: Materials and Methods

1. Case selection

This project involved two lines of investigation. The first was a retrospective analyses of the tumour microenvironment from archived samples. The second was

a prospective collection of fresh blood samples to assess the proportion of Treg in FCR with HS. For the retrospective part of this study histological samples were selected from the pathology database of the Department of Veterinary Medicine, University of Cambridge. A total of 27 cases were selected from formalin fixed and paraffin-embedded (FFPE) tissue samples collected between 2000 and 2017. All cases had a confirmed diagnosis of HS (CD18+ and MHC class II+), staining for CD18 was not repeated for this project. All tumours were localized in soft tissue and belonged to the histopathological histiocytic subgroup (Fig 2.1). Fresh frozen samples from 3 canine HS were used for validation of the antibodies (28, FC 95/35; 29, FC 96/14 and 30, FC 97/92).

A B

Figure 2.1 Haematoxylin and eosin staining of a histiocytic-type HS. A (20x magnification, 100 µm scale bar), B (40x magnification, 50 µm scale bar).

For the prospective part of the study, cases were collected amongst the patients referred to the oncology team at the Queen’s Veterinary School Hospital, University of Cambridge and local clinics during the time of this study.

Chapter 2: Materials and Methods

2. Sample collection

Histological samples of the tumours were collected by the pathology team at the Department of Veterinary Medicine during post-mortem examination, or as biopsies by clinicians as part of work-up at the Queen’s Veterinary Hospital, University of Cambridge. Owners consent was obtained before the post-mortem examination or diagnostic procedure. The only case from 2017 was sent in formalin by a first opinion veterinary surgeon. In this case the client signed a written consent form to allow donation of the tissue specimen. Canine tissue collected from unaffected organs during routine post-mortem examination at the pathology section of the Department of Veterinary Medicine was used as control.

The peripheral blood samples were collected in 2.5 ml EDTA tubes by the oncology team at the Queen’s Veterinary Hospital, University of Cambridge, as part of the diagnostic work-up. Owners signed consent to allow the use of excess blood for research (Appendix 1). Veterinary surgeons from different practices also sent EDTA blood samples accompanied by animal history, confirming diagnosis of histiocytic sarcoma, and a signed consent form from the owner. The control group included FCR over the age of 7 that had not been diagnosed with any form of cancer. Owners donated residual volumes of blood from samples collected as part of general health screens to the research project. Following collection contact was maintained with the owners to ensure the dogs did not go on to develop HS during the time of the study.

The research proposed was reviewed by the Department of Veterinary Medicine Ethics and Welfare Committee and approval was given before samples were collected (CR164).

3. Histopathology

3.1 Preparation and staining All samples were fixed in 10% neutral-buffered formalin solution at room temperature and embedded in blocks of paraffin wax. 5 µm sections were cut from the wax embedded tissue and mounted on positively charged microscopy slides (Snowcoat; Surgipath Europe Ltd., Peterborough,UK). Sections were dried

Chapter 2: Materials and Methods

overnight at 50 °C. Staining with haematoxylin and eosin was performed on the Leica ST4040 linear staining system. Tumour samples and control samples underwent the same treatment. Slides were evaluated by light microscopy by one board certified pathologist. Samples with excessive areas of necrosis were excluded from the study.

4. Immunohistochemistry

4.1 Antibody validation Immunohistochemistry was performed with immunolabeling for CD3 (mouse anti- human, F7-2-38, Dako), MHC class II (mouse anti-human, TAL.1B5, Dako), E- cadherin (mouse anti-human, NHC-38, Dako), FoxP3 (rabbit anti-human polyclonal, Spring Bioscence), IL-10 (rabbit anti-dog polyclonal, GeneTex) and PD-L1 (rabbit polyclonal, Novus). Negative controls were isotype monoclonal or species polyclonal matched. Purified mouse IgG1, κ isotype (MOPC-21, Biolegend) and rabbit immunoglobulin G (Sigma) were used at the same concentration as anti-marker antibodies. Appropriate antibody dilutions were determined by titration of each antibody on formalin-fixed paraffin-embedded canine tissue (see Table 2.1). The secondary antibody used was an anti- rabbit/mouse (FLEX HRP, Dako).

Marker Dilution Stock Isotype control Control tissue Concentration section (ug/ml) CD3 1:150 138 mouse IgG1 lymph node, spleen

MHC-II 1:400 24 mouse IgG1 lymph node, spleen

E-cadherin 1:200 55.7 mouse IgG1 lymph node, spleen

FoxP3 1:500 370 rabbit IgG lymph node, thymus

IL-10 1:300 500 rabbit IgG spleen, intestine

PD-L1 1:150 1000 rabbit IgG spleen

Table 2.1 Summary of antibodies used for immunohistochemistry.

Chapter 2: Materials and Methods

4.2 Antigen Retrieval Antigen retrieval was heat mediated for the majority of antibodies. Sections were dried overnight at 50 °C and then treated for deparaffinization, rehydration, and antigen-retrieval in a combined 3-in-1 procedure using the PT link Envision Flex antigen retrieval solution, in a high pH buffer (Dako, Carpinteria, CA, USA). Sections were immersed in a preheated solution at 65 °C and pH 9.0. Once the solution had reached the working temperature of 97 °C, slides were incubated for 20 minutes, then allowed to cool down and rinsed in buffer (Dako Envision wash buffer) at room temperature. Enzymatic antigen retrieval was performed for PD-L1 staining, to improve epitope unmasking. Slides were de-paraffinised in two changes of xylene, hydrated through graded alcohols, and then rinsed in distilled water. Sections were then covered with Proteinase K (Dako) for 8 minutes at room temperature. 4.3 Staining Staining was performed using an automated slide processing system (Autostainer plus Link, Dako). The first step is the blocking of endogenous peroxidase, this was performed by treating the slides with Flex HP block for 30 minutes. Non-specific binding of the primary antibody was blocked with 10 % serum of the host species of the secondary antibody for 30 minutes and 1:100 of the same serum in the preparation of the primary antibody. The tissue was then covered with the primary antibody for 1 hour at room temperature. Following a wash with EnVisionTM FLEX Wash Buffer, the tissue was incubated with the secondary antibody for 30 minutes (FLEX HRP, Dako). Horseradish peroxidase (HRP) was used as the enzymatic reporter label for all samples. For visualisation, the sections were incubated with 3,3'-diaminobenzidine solution (DAB+, Dako) for 5 minutes and then counterstained with Mayer's hematoxylin. 4.4 Scoring Labelling was scored semi-quantitatively. The mean percentage of labelled cells for each sample was assessed in 10 randomly selected high-power magnification fields (x40). If 10 high-power fields were not present in a specimen then all of the tissue was examined. The scoring was: 1, for 1 % to 20 % positive cells; 2, for 21 % to 40 % positive cells; 3, for 41 % to 60 % positive cells; 4, for 61 % to 80 % positive

Chapter 2: Materials and Methods

cells; 5, 81 % to 100 % positive cells. If no staining was present the sample was described as negative and scored 0. Samples were also assessed for intensity of staining. Signal was described as “strong” when comparable to intensity of positive control, it was described as “mild” when barely visible, and as “moderate” when intensity of staining was between the two extremes.

4.5 Imaging All samples were evaluated by light microscopy and scanned into digital images via a Nanozoomer slide scanner (Hamamatsu®), but no image analysis was performed due to insufficient samples for machine-teaching.

5 Immunofluorescence

5.1 Sample preparation A Leica CM 1900 cryostat was used to cryosection 20 µm thick sections of three frozen samples of HS. OCT was used as embedding medium for the frozen tissue specimens. The sections were placed on slides and left to dry at room temperature for 4 hours. Tissue sections were then fixed in 2 % paraformaldehyde (PFA) for 10 minutes at room temperature. 5.2 Antibody validation Table 2.2 shows the antibodies against PD-L1 (CD274) tested during optimization of the immunofluorescence protocol. Rabbit polyclonal anti-CD274 from Novus was selected as the best performing antibody and used on three frozen canine HS samples at a dilution of 1:100 (stock concentration 1mg/ml).

Secondary Control tissue Primary Antibody Dilution Isotype Antibody section Ant-human CD274 1:100 Rb Goat Anti-Rabbit Human intestine (Novus) Polyclonal AlexFluor 488

Anti-human CD274 1:100 Rb Goat Anti-Rabbit Human intestine

(ThermoFisher) Polyclonal AlexFluor 488

Anti-human CD274 1:100 Mouse Goat Anti-Mouse Human intestine (M1H1, Biolegend) IgG2a AlexFluor 488

Table 2.2 Antibodies tested for reactivity to canine PD-L1 by immunofluorescence.

Chapter 2: Materials and Methods

5.3 Staining Following fixation, slides underwent three washes of 3 minutes each in phosphate- buffered saline (PBS). Samples were then covered in blocking agent (10 % goat serum, 2 % bovine serum albumin, 0.3 M glycine in PBS) for 1 hour at room temperature. Primary antibodies were then added and sections were incubated overnight at 4 ºC. The slides were then washed five times in PBS, each time for 3 minutes. The secondary antibody was added (dilution of 1:400) and sections incubated for 1 hour at room temperature, followed by three 3 minute washes in PBS. Cell nuclei were counterstained with ToPro-3 nuclear stain (1 µM, Thermofisher) for 20 minutes at room temperature in the dark. After three more 3 minute washes in PBS, sections were mounted in ProLong Gold anti-fade mountant (Thermofisher). Slides were kept in the dark until imaging.

5.4 Imaging Images were collected using an SP5 confocal microscope.

6 Flow Cytometry

6.1 Antibodies Cells were stained with anti-dog CD3:FITC, CD4:RPE, CD8:Alexa Fluor® 647 (CA17.2A12, YKIX302.9, YCATE55.9, TC023 Bio-Rad, 1:13 dilution) for the detection of T cells. Isotypes used were respectively mouse IgG1, rat IgG2a and rat

IgG1 conjugated to the relative fluorophore. To identify Treg cells, the events were stained with rat anti-dog CD4:FITC (YKIX302.9, Bio-Rad, 1:10 dilution), mouse anti-dog CD25:efluor 660 (P4A10, eBioscience, 1:20 dilution) and rat anti-dog FoxP3:PE (FJK-16s, eBioscience, 1:20 dilution), an intracellular antibody. Isotypes used were rat IgG2a:FITC negative control (Bio-Rad), mouse IgG1:efluor660 (P3.6.2.8.1, eBioscience) and rat IgG2a, :PE (eBR2a, eBioscience). The most recent samples were also stained with anti-CD8:FITC (YCATE55.9, eBioscience), anti-CD25:efluor 660 and anti-FoxP3:PE in a separate panel to allow identification

of induced Treg cells. The different panels of antibodies are shown in Table 2.3.

Chapter 2: Materials and Methods

Accuri C6 FL1 FL2 FL3 FL4 FITC PE PerCp Alexa -660/700

laser (nm) 488 488 488 640 Filter (nm) 530/15 585/20 670 LP 675/12.5 T cell panel

Kit anti- CD3- FITC Kit anti-CD8 Alexa Kit anti- CD4-PE

Treg panel

Rat anti-dog CD4 (or CD8)-FITC

Mouse anti- dog CD25- eFluor 660 anti - mouse/rat FoxP3-PE

Table 2.3 Summary of antibodies used for flow cytometry. T cells are defined as CD3+CD4+ or CD3+CD8+.

+ + + Treg are defined as CD4 CD25 FoxP3 .

Chapter 2: Materials and Methods

6.2 Sample preparation Peripheral blood samples were received in 2.5 ml EDTA tubes and processed within 24 hours from collection. Lymphocytes and mononuclear cells were separated from red cells by layering the blood on 3 ml of Histopaque® – 1077 (Merck) at room temperature and centrifuged for 30 minutes at 400 x g. The interface cells were then washed 3 times in PBA (500 ml of PBS, 0.5 g of bovine serum albumin, stored at 4 ºC). The concentration of the cells recovered was measured on a SYSMEX analyser. The prepared sample was divided into tubes, each containing 106 cells, and the cells were incubated with the antibodies for 60 minutes in the dark at room temperature. Following incubation, ammonium chloride was added to the sample to lyse remaining red blood cells. PBS was used for subsequent washes.

For intracellular staining, surface stained cells (anti-CD4 or anti-CD8 and anti- CD25) were incubated for 60 minutes at 4 ºC in the fixation and permeabilization solution from the FIX & PERM Cell Fixation & Cell Permeabilization Kit (Thermofisher). The following washes were performed with the Permeabilisation Buffer Solution from the same kit. The intracellular antibody (anti-FoxP3) and the correct isotype were then added to the fixed cells diluted in the permeabilisation buffer and incubated in the dark at room temperature overnight. The samples were washed twice in permeabilisation buffer solution and suspended in PBA for analysis.

6.3 Flow cytometry analysis Before analysis, colour compensation was set using compensation beads (OneCompTM eBeads Compensation Beads, Invitrogen, Thermo Fisher Scientific) following the manufacturer’s protocol.

Data were collected using a first gate set on the lymphocyte population based on typical forward and side scatter characteristics in which 10,000 events were collected. This was confirmed during analysis by back gating on CD3 positive cells (data not shown). For cells stained with the T cell panel, the proportion of CD3+CD4+ cells [FL1/FL2], CD3+CD8+ cells [FL1/FL4] and CD3+CD4-CD8- was

determined (Figure 2.2). For the Treg panel the lymphocyte FSC/SSC profile was moved to account for cell shrinkage caused by fixation. Again, 10,000 events were collected in the gate. During analysis, positive CD4 cells were identified

Chapter 2: Materials and Methods

[FL1/SSC] and within this subgroup the cells also positive for CD25 and FoxP3 [FL4/FL2] were then characterized (Fig 2.3). When assessing CD8 positive cells the same procedure was followed.

1 2 3

4 5

Figure 2.2 T cell flow cytometry profile.

Example of a flow cytometry profile for T cell surface markers. Panel 1: lymphocyte gate; Panel 2: CD3 and CD4 isotype control of lymphocytes; Panel 3: CD3 and CD8 isotype control of lymphocytes; Panel 4: CD3 and CD4 staining of lymphocytes, the red circle shows events positive for both CD3 and CD4; Panel 5: CD3 and CD8 staining of lymphocytes, the red circle shows events positive for CD3 and CD8.

Chapter 2: Materials and Methods

1 2 3

4 5

Figure 2.3 Treg flow cytometry profile.

Example of a flow cytometry profile for the Treg markers. Panel 1: lymphocyte gate of fixed cells; Panel 2: the isotype control for CD4 staining showing the CD4 positive gate; Panel 3: lymphocyte CD4 staining profile showing the CD4 positive gate; Panel 4: the isotype control for CD25 and FoxP3 on CD4+ lymphocytes is shown; Panel 5: CD25 and FoxP3 staining of CD4+ lymphocytes. The purple circle highlights events positive for FoxP3; the orange circle highlights events positive for CD25; and the red circle highlights events positive for both CD25 and FoxP3.

Chapter 2: Materials and Methods

7 Reverse transcription PCR

7.1 Sample preparation Tissue sections from FFPE non-affected canine spleen and sub-cutaneous HS were cut into 10 µm thick sections, excess paraffin was removed with a surgical blade. Sections were placed in a 1.5 ml microcentrifuge tube and processed as follows. 7.2 Purification of total RNA from FFPE tissue Deparaffinization and purification was performed using the RNeasy FFPE kit (Qiagen®) following the manufacturer’s protocol. Briefly, paraffin was removed using deparaffinization solution (Qiagen®). Samples were incubated with proteinase K to release RNA from the sections. Subsequently heat was used to partially reverse formalin crosslinks. The samples was then treated with DNase to eliminate all genomic DNA and applied to an RNeasy MinElute spin column (Qiagen®), where RNA bound to the silica membrane. RNA was then eluted in 30 µl of RNase-free water. 7.3 Primer selection Primers for reference genes were selected from the literature (Table 2.4). Those for PD-L1 and PD-1 were designed for the project. The canine PD-L1 (AB898678) and PD-1 (AB898677) mRNA sequences were retrieved from GenBank and compared to human PD-L1 (NM_014143) and PD-1 (NM_005018) mRNA sequences to infer intron and exon boundaries. Primers were designed using Primer3 (http://primer3.ut.ee), limiting the position of the forward and reverse primers to encompass an exon - exon boundary (Table 2.4).

Chapter 2: Materials and Methods

NAME SEQUENCE Reference Ta GUSB-F1 AGACGCTTCCARTACCCC Brinkhof et al., 62 ºC 2006 GUSB-R1 AGGTGTGGTGTAGAGGAGCAC 62 ºC HPRT1-F1 ASTTGCTGGTGAAAAGGAC Brinkhof et al., 56 ºC 2006 HPRT1-R1 TTATAGTCAAGGGCATATCC 56 ºC RPL8-F1 CCATGAAYCTGTGGAGC Brinkhof et al., 55 ºC 2006 RPL8-R1 GTAGAGGGTTTGCCGATG 55 ºC RPL13A-F1 CTGCCCCACAAGACCAAG Clements et al., 56 ºC 2006 RPL13A-R1 GGGATCCCATCAAACACCT 56 ºC RPS5-F1 TCACTGGTGARACCCCCT Brinkhof et al., 62 ºC 2006 RPS5-R1 CCTGATTCACACGGCGTAG 62 ºC RPS19-F1 CCTTCCTCAAAARTCTGGG Brinkhof et al., 61 ºC 2006 RPS19-R1 GTTCTCATCGTAGGGAGCAAG 61 ºC caPD1-F1 CCCCAACACACAGATCAACG 442-461 59 ºC caPD1-R1 GAAGACAGCGGCCAGGAC 637-620 59 ºC e2 – e3 196 bp caPD-L1-F1 TCTTGGTGTAGTCCTGGCAG 819-838 59 ºC CaPD-L1-R1 CTGAACTCAAACCACAGGCC 1004-985 59 ºC e5-e7 186 bp

Table 2.4 Primers used for RT-PCR (e is human exon number).

7.4 Reverse transcription of RNA SuperScript® IV first-strand cDNA synthesis reaction (Thermofisher) master mix was prepared following manufacture’s procedure. To anneal primer to template RNA 13 µl of master mix composed of 2.5 µM of random hexamers, 0.5 mM an aqueous solution of dATP, dCTP, dGTP (dNTP), 500 ng of template RNA and nuclease-free water were mixed and briefly centrifuged. The RNA-primer mix was

Chapter 2: Materials and Methods

heated at 65 ºC for 5 minutes and then incubated on ice for approximately 2 minutes. During incubation an RT reaction mix was prepared with 4 µl SuperScript® IV buffer, 1µl of 100mM DTT, 1µl of RNaseOUTTM recombinant RNase inhibitor and 1 µl of SuperScript® IV reverse transcriptase (200 U/µL). This RT reaction mix was then added to the annealed RNA and incubated at 23 ºC for 10 minutes, and then for 10 minutes at 55 ºC. The reaction was then inactivated at 80 ºC for 10 minutes. The cDNA product was then stored at -80 ºC before PCR amplification.

7.5 RT-PCR conditions The KAPA SYBR FAST qPCR master mix kit (KapaBiosystems®) was used in a trial RT-qPCR on the deparaffinised samples as per the manufacturer's protocol. Four dilutions of starting template of purified RNA were trialled, starting from 50 ng RNA per reaction, then 10 ng, 2 ng and 0.4 ng. Master mix was prepared as per the manufacturer's instructions and a no-template control (NTC) was included to enable detection of contamination of reaction components, while a no-RT control was included to enable detection of contaminating genomic DNA. Transfer RNA was used as a diluent, as it would not be amplified by the primers. All samples, NTC and no-RT control were run in triplicate using the same cycling conditions for all assays (Table 2.5). The no-RT sample had not received SuperScript® IV reverse transcriptase. After data were collected, samples with primer dimer, as assessed by the melt curve analysis, were excluded from analysis.

1 sec at 20 sec 72 ºC 3 sec at at Ta 3 min at 95 ºC 95 ºC

Table 2.5 Cycling conditions. Used for second step of two-step RT-PCR reactions. Highlighted steps were repeated 40 times for each assay. Ta represents annealing temperature as defined in Table 2.4.

Chapter 2: Materials and Methods

8 Submarine Agarose Gels

PCR products were analysed by agarose gel electrophoresis on 2 % agarose gel with 2.5 µg ethidium bromide/ml. Tris-Acetate-EDTA buffer was used. An Invitrogen 100 base pair ladder was used for the marker track. 5 % glycerol: Bromophenol blue loading buffer was added to the samples before loading in gel microwells. The gel was run at 100 V, 250 mA for 25 minutes. The gel was visualised on a UV transilluminator glass and an image was taken before bands were cut out to extract DNA.

9 DNA purification

DNA was purified from agarose gel fragments following the protocol for Wizard® SV Gel and PCR Clean-Up System (Promega). Briefly, the gel slice was placed in a 1.5 ml microcentrifuge tube, 10 µl of membrane binding solution was added to every 10 mg of agarose. The tube was incubated at 65 ºC until the gel slice had completely dissolved. The gel mixture was then transferred to a mini-column and following incubation at room temperature for 1 minute, it was centrifuged at 16,000 × g for 1 minute. The flow through was placed on the column a second time to increase yield. 700 µl of membrane wash solution, containing ethanol, was used to wash the sample twice, the flow through was discarded each time. DNA was eluted with 30 µl of nuclease-free water (10 µl less than suggested in protocol) because of the low amounts of DNA in fragments. This provided the concentration of DNA required for sequencing.

10 Sequencing

Sequencing was commissioned by Source Bioscience (Sanger sequencing service). The results of sequencing were analysed by using the online nucleotide Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) tool, against the non-human and non- mouse nucleotide database. If this did not show homology to nucleotides in the

Chapter 2: Materials and Methods

database, EMBOSS explorer matcher was used to perform a local alignment against the specific canine gene sequences.

11 Statistics

Statistical analysis of flow cytometry results was performed using GraphPad Prism 5. Normality of groups was assessed by the Shapiro-Wilk normality test (the group size of the tumour affected dogs was too small for the D'Agostino and Pearson omnibus normality test). As some of the groups were not normally distributed, group comparisons were performed using a non-parametric Mann-Whitney U test. Statistical significance was taken as p<0.05. Power analyses were performed using the sample size calculator (https://www.stat.ubc.ca/~rollin/stats/ssize/n2.html) using 0.05 significance levels with 80% power.

Chapter 3: Results

1. Histopathology

In total 27 cases of histiocytic sarcoma were selected from archived tissue, all samples were from localised tumours. Sections stained with hematoxylin and eosin (H&E) were reviewed to assess the distribution, pattern and cell morphology of neoplastic cells. For each tumour, cells were assessed on 20 random fields at 20x magnification where neoplastic tissue was present. Areas of necrosis and haemorrhage were excluded. Based on the histological presentation, tumours were classified as belonging to the histiocytic subgroup (Fig 3.1). Only samples where over half of the neoplastic cells belonged to the histopathological histiocytic subgroup were included in the study (Appendix 2) (Constantino-Casas et al., 2011). The samples showed sheets of round and polygonal cells, occasional multinucleate giant cells. High levels of mitotic activity were recorded and occasional bizarre mitoses were observed. Cells were generally large, with abundant cytoplasm; cytoplasmic vacuolization was occasionally observed. Nuclei were generally oval and anisokaryosis was noticed. Nucleoli were increased in size and multiple nucleoli were detected. Necrosis was evident in some samples.

No sex predisposition was noticed within the cases collected (14 male and 13 female). The collected tumours originated from FCR ranging in age from 6 to 11 years, mean age at time of diagnosis was 8.2 years. The majority of the tumours were localised on limbs. 19 (70.4%) tumours were located on the front limb, 5 (18.5%) were located on the hind limb, 1 (3.7%) on the dorsum, 1 (3.7%) on the flank, and 1 (3.7%) in the groin.

37 Chapter 3: Results

Figure 3.1 Hematoxylin and eosin staining of tumour 21. A 20x (100 µm scale bar) and B 40x (50 µm scale bar) magnification respectively. At lower magnification the densely arranged sheets of neoplastic cells can be appreciated. At higher magnification a mitotic figure and vacuolisation of cytoplasm are noticeable.

38 Chapter 3: Results

2. Immunohistochemistry

One of the aims of this project was to investigate possible immunomodulatory molecule expression within the tumour microenvironment. To do this immunohistochemistry was used with a panel of antibodies. For all antibodies that showed reactivity, immunopositive labelling was present in the positive control and not detected in the negative (isotype) control. MHC class II expression is known to be high on the tumour cells (Kato et al., 2013) and this was confirmed with high level of staining detected for MHC class II, with 9 of the tumours scoring 5/5; on average the slides were scored as 4 expressing MHC class II in over 60% of cells (Table 3.1). Staining was described as strong and observed in the cytoplasm and membrane (Fig 3.2). E-cadherin is a marker of Langerhans cells and HS tumour cells should be negative for this marker (Moore, 2014). No staining for E-cadherin was detected in any of the 27 tumours. On the positive control staining was found in cytoplasm and membrane (Fig 3.2, Table 3.1). CD3 was used to detect infiltrating T cells and staining was found in both the membrane and cytoplasm (Fig 3.3). Staining for this marker was strong in intensity and an average score of 1/5 was given to all samples (Table 3.1). Previous work (Marcinowska et al., 2017) has shown that most of the infiltrating T cells are FoxP3 positive. This was also true in this study, with similar frequency of FoxP3, and CD3+ cells. FoxP3 staining was always nuclear and all positive tumours scored 1 (2 tumours were negative for CD3 and FoxP3), intensity of staining was always strong (Fig

3.3, Table 3.1). One of the mechanisms used by Treg cells to induce immune suppression is secretion of IL-10. Although the antibody used could be shown to label the positive control section, there was little staining of the HS tumours. Staining for IL-10 was negative on almost all samples. On the 3 samples that showed weak immunopositivity (Table 3.1), staining was cytoplasmic and granular in appearance. Whereas it was dark and nuclear in the positive control (Fig 3.4).

To further interrogate the tumour microenvironment staining was attempted with anti- human antibodies for IDO (goat polyclonal, Abcam), PD-1 (rabbit monoclonal, Abcam), perforin (dG9, Biolegend) and granzyme B/H (rabbit polyclonal, Abbexa). All tissue used was formalin fixed and paraffin-embedded. Samples of non-diseased spleen, thymus, lymph node and intestine were used as positive controls and immunolabelling of tumour

39 Chapter 3: Results tissue was also attempted. During antibody validation slides underwent heat-induced (low pH and high pH buffer) and enzymatic antigen retrieval. Multiple dilutions of the primary antibody were incubated for 60 min at room temperature and at 4 ºC overnight, to allow for lower affinity binding. No specific staining was achieved.

40 Chapter 3: Results

A B

C D

E F

Figure 3.2 Immunohistochemical staining for MHC class II and E-cadherin. MHC class II staining at 40x magnification (50 µm scale bar) A, positive control on spleen, B tumour, C matched isotype. E-cadherin staining at 40x magnification (50 µm scale bar) D, positive control on canine histiocytoma, E tumour, F matched isotype.

41 Chapter 3: Results

A B

C D

E F

Figure 3.3 Immunohistochemical staining for CD3 and FoxP3. CD3 staining at 40x magnification (50 µm scale bar) A positive control on thymus, B tumour, C matched isotype. FoxP3 staining at 40x magnification (50 µm scale bar) D positive control on thymus, E tumour, F matched isotype.

Chapter 3: Results

A B

Figure 3.4 Immunohistochemical staining for IL-10. Images at 40x magnification (50 µm scale bar) A positive control on intestine (serosa), B tumour, C matched isotype.

43 Chapter 3: Results

Case Number CD3 MHC II FOXP3 E- IL-10 PD-L1 cadherin 1 1 2 1 0 0 3 2 1 2 1 0 0 2 3 1 3 1 0 0 1 4 0 4 0 0 0 4 5 1 5 1 0 0 4 6 1 5 1 0 0 4 7 0 1 0 0 0 5 8 1 5 1 0 0 4 9 1 5 1 0 0 2 10 1 5 1 0 1 x 11 1 5 1 0 0 5 12 1 5 1 0 0 4 13 1 2 1 0 0 5 14 1 5 1 0 0 1 15 1 2 1 0 0 2 16 1 1 1 0 1 3 17 1 4 1 0 0 4 18 2 5 1 0 0 3 19 1 4 1 0 1 3 20 2 3 1 0 0 5 21 3 5 1 0 0 4 22 1 4 1 0 0 1 23 1 2 1 0 0 4 24 1 3 1 0 0 5 25 1 2 1 0 0 1 26 1 2 1 0 0 3 27 1 3 1 0 0 5

Table 3.1 Individual scoring for each tumour with the markers shown. 1, 1-20% of positive cells; 2, 21-40% of positive cells; 3, 41-60% of positive cells; 4, 61-80% of positive cells; 5, 81-100% of positive cells.

44 Chapter 3: Results

3. Flow Cytometry

To investigate if the upregulation of Treg was a localised phenomenon only or reflected systemic changes in this population of cells eight dogs with histiocytic sarcoma and 11 clinically healthy controls were enrolled in this prospective study (Appendix 3). Peripheral blood was collected and analysed with the antibody panels previously

described, one of the affected samples was only labelled with the Treg cells panel.

The median age at time of diagnosis was 8y (5y9m to 10y6m) and 4 (50 %) dogs presented with metastasis at time of diagnosis. The majority of the samples were collected from dogs receiving medical palliative treatment only (non-steroidal anti- inflammatories), 2 dogs also received palliative radiotherapy, for 1 of these it was in conjunction with lomustine. One case had undergone amputation.

There was a trend to a significant difference between the groups in the proportion of CD3+ lymphocytes (Table 3.2, Fig 3.5); power analysis suggested that group sizes of 15 would be needed to see a significant difference. However a difference was recorded between the CD4/CD8 T cell ratio, this being higher for the affected dogs. This was driven by the frequency of CD3+CD8+ T cells which was higher in the control group (Table 3.2, Fig 3.5). Within the affected group, a higher frequency of CD4-CD8- T cells

(possibly γδ T cells) was also recorded (Table 3.2, Fig 3.5). With the Treg antibody + + + panel, no differences were found between populations of CD4 CD25 FoxP3 Treg in

affected and healthy dogs (Table 3.3, Fig 3.6). Treg/CD8 ratio was assessed (data not shown) and no difference was found (Fig 3.6). Power analyses on the results from the cell types that did not show a significant difference between groups suggest that group sizes of >37 would be needed to see significant differences.

45 Chapter 3: Results

% CD3+ % within CD3+ population CD4/CD8 ratio

CD4+ CD8+ CD4-CD8-

Affected Mean 46.9 48.9 26.7* 24.4** 2.4*

Standard 22.5 9.4 13.2 13.7 1.3 deviation

Control Mean 67.5 48.8 40.5 11.5 1.2

Standard 13.9 4.5 6.1 3.5 0.3 deviation

Table 3.2: Summary of results from T cell surface markers in peripheral blood. Statistically significant differences between affected and control groups were determined by a Mann-Whitney U test; * p<0.05, **p<0,01.

% within CD4+ population

CD25+ FoxP3+ CD25+FoxP3+

Affected Mean 3.1 6.8 0.6

Standard deviation 4.2 4.5 0.6

Control Mean 5.6 4.6 1.0

Standard deviation 6.3 3.5 0.7

Table 3.3: Summary of results from Treg markers in peripheral blood. No statistically significant differences between affected and control groups were found.

46 Chapter 3: Results

Figure 3.5 Peripheral blood lymphocyte surface marker expression. The graphs show means and 95% confidence intervals. Statistically significant differences between affected and control groups were determined by a Mann-Whitney U test; * p<0.05, **p<0,01.

47 Chapter 3: Results

Figure 3.6 Peripheral blood Treg marker expression. The graphs show means and 95% confidence intervals. No statistically significant differences between affected and control groups were found.

48 Chapter 3: Results

4. PD-L1 antibody validation 4.1 Immunofluorescence

Difficulties with antigen retrieval on FFPE tissue are often present, so to screen possible canine cross-reactive antibodies against PD-L1, sections from three frozen tumour samples were used with 3 different antibodies (see Chapter 2). Only one antibody showed apparent cross-reactivity with canine tissue, the rabbit anti-human CD274 antibody from Novus (Figure 3.7).

All 3 tumours showed positive immunofluorescence for PD-L1, although the amount of signal varied between tumours. Tumour 28 showed the weakest staining with low intensity fluorescence and positive cells being in certain regions of the tumour; tumour 29 showed middle levels of staining intensity but all tumour cells appeared to be positive; tumour 30 had the highest intensity of staining in all tumour cells (Fig 3.7). This allowed identification of an anti-human PD-L1 antibody that apparently cross- reacted with canine PD-L1. However cross species reactivity may sometimes be to spurious proteins. To confirm expression of PD-L1 RT-PCR was used to determine if PD-L1 mRNA could be detected in tumours.

49 Chapter 3: Results

A B

C D

E F

Figure 3.7 Immunofluorescent staining for PD-L1. A,B tumour 28. C, D tumour 29. E, F tumour 30. Confocal micrographs taken with a 63x objective (50 µm scale bar). For each tumour the left hand panel shows the isotype control whilst the right hand panel shows the antibody staining. Within each panel of

50 Chapter 3: Results images shown on the top are all colour channels unthresholded to the left and all channels thresholded, based on isotype signal across all animals, on the right. On the bottom there is thresholded signal to the left and actin stain to the right. Courtesy of Dr J Robertson.

4.2 Reverse transcription PCR

RT-PCR may be used to investigate gene expression in samples, however to do this quantitatively each sample must have its gene of interest expression level normalised to reference gene expression levels. MIQE guidelines suggest at least 2-3 reference genes be used. The literature was interrogated for canine reference gene RT-qPCR assays. Assays from Brinkhoff et al. (2006) were selected as these were based on SYBR assays and the 5 reference gene assays showing the best stability across different tissues and cell types (Brinkhof et al., 2006) including skin and atopic dermatitis (Schlotter et al., 2009) were used. To increase the number of assays to 6, a last one was chosen from Clements et al. (Clements et al., 2006; Ayers et al., 2007) that was stable across cartilage and synovial fluid. However, the assays from this paper were locked nucleic acid fluorescence resonance energy transfer probe assays and so had less likelihood of working specifically with the SYBR assays I wanted to perform.

For PD-1 and PD-L1, the canine mRNA sequences were taken from GenBank by searching the Nucleotide-NCBI database. PD-1 searches gave 2 sequences (AB898677.1 and NM_001314097.1) which were 99.9 % identical (the difference was 918 T:909 C respectively). The longer sequence (AB898677.1) was used to design primers. This was aligned against the human PD-1 sequence (NM_005018.2) (Appendix 4) and the predicted exon-exon boundaries noted. Primers were designed across the exon 2-3 boundary.

A canine PD-L1 search in Nucleotide-NCBI gave 2 sequences (AB898678.1 and NM_001291972.1) which were 100% identical. By using this to BLAST search, 8 different predicted transcript variants were seen (X1-X8) in GenBank. Human PD-L1 mRNA has 4 transcript variants in GenBank. The 13 sequences were aligned and the output of the alignment (Appendix 5) was marked for the position of the exons and start and stop codons from the first human transcript variant sequence (NM_014143.3) and the start and stop

51 Chapter 3: Results codons of the canine sequence (AB898678.1). Primers were designed across the exon 5-7 boundaries.

Initially to investigate the use of RT-PCR to measure RNA expression in the tumours, RNA was extracted from one tumour and one unaffected spleen from paraffin blocks using the Qiagen RNeasy FFPE extraction kit. Trial RT-PCRs were performed with a one-step protocol using the KAPA SYBR FAST One-Step qRT-PCR master mix kit. Results showed a high level of primer dimers (data not shown) on assays with GUSB and RPS5. Although specific product from GUSB was purified (sequence not shown), a two-step protocol was investigated to reduce this primer dimer background. This and subsequent PCR reactions were performed by first making a cDNA using random hexamers and then using the KAPA SYBR FAST qPCR kit. These reactions were cleaner by melt curve analysis. The curves are shown for each gene tested (Fig 3.8 – 3.11). The curves all showed major peaks >75 °C but there was often a tail at lower temperatures with a peak at approximately 60 °C which suggested that primer dimers were still present. In some assays there were also occasional products that were suggestive of mispriming products being present in the assay (eg Fig 3.11A, RPS19). HPRT-1 showed probable products in the spleen and tumour samples that had different melt temperatures (Fig 3.11B).

To determine if the PCR products were the correct size, they were run on agarose gels (Fig 3.12). The assays all produced bands of predicted size, however primer dimers and other probable mispriming products were also present. Specific bands were purified and sent for sequencing. The amplicons were determined to be correct for the PD-1, PD-L1, GUSB, RPS5, RPS19 and HPRT-1 assays by alignment to database sequence (Appendix 6). The HPRT-1 sequence was derived from spleen RNA. The sequences were not 100% identical to the database sequences. Some of the mismatches may be due to poor sequence quality as the PCR products were very short. However, looking at some of the errors in the sequence, there is a common fault where the primers had degeneracy (eg. C/G) positions. All of these have missing bases compared to the canine database sequence suggesting a transcription error into the Brinkhoff et al. (2006) paper. The HPRT1 forward primer did not match the 5’ base and by further analyses of mRNA variants and expressed sequence tags that these identified by Blast (Appendix 7) it could be seen that again it was probably a fault of the primer degeneracy when copying into the paper. The HPRT-1 sequence of

52 Chapter 3: Results

PCR product from tumour was not obtained fully and so could not be fully compared against the PCR product from spleen. I therefore do not know if the difference in melt temperature from the two samples was due to sequence variation. However the agarose gel did not show an obvious difference in size in the products from spleen or tumour (Fig 3.12). RPL8 and RPL13A products gave poor sequencing quality. These assays were excluded from further analyses. This therefore gave 4 possible reference gene assays to use together with the genes of interest, PD-1 and PD-L1. The assays will need further optimisation to be able to be used quantitatively due to the background seen. However I ran the assays on 4 tumour samples to determine if PD-L1 or PD-1 mRNA could be detected. Samples were considered PCR positive when they displayed fluorescence signal above background threshold and generated well-defined, temperature specific peaks in the melting curve analysis. The PCR analyses allowed validation of expression of the PD-L1 gene within the tissue of interest. PD-1 gene expression was not detected in tumour samples, although this was high on the sample of spleen (Table 3.4).

Gene Ct value

Tumour I Tumour II Tumour III Tumour IV Spleen

Genes of interest

PD-1 ND ND ND ND 31.93

PD-L1 26.97 33.91 30.75 34.39 ND

Housekeeping genes

GUSB >40 >40 37.84 >40 24.90

HPRT1 29.39 36.61 32.79 36.09 x

RPS5 >40 >40 34.98 >40 26.64

RPS19 37.76 38.86 35.06 36.65 x

Table 3.4 Cycle threshold (Ct) values for assays performed. cDNA in 1/5 dilution.

53 Chapter 3: Results

A B

Figure 3.8 Melt curves for PD-L1 and PD-1 RT-qPCR assays. A PD-L1, B PD-1. Legend: RED - no template control, GREEN – spleen cDNA, BLUE – tumour cDNA.

54 Chapter 3: Results

A B

Figure 3.9 Melt curves for GUSB and RPL8 RT-qPCR assays. A GUSB, B RPL8 Legend: RED - no template control, GREEN – spleen cDNA, BLUE – tumour cDNA.

55 Chapter 3: Results

A B

Figure 3.10 Melt curves for RPL13A and RPS5 RT-qPCR assays. A RPL13A, B RPS5. Legend: RED - no template control, GREEN – spleen cDNA, BLUE – tumour cDNA.

56 Chapter 3: Results

A B

Figure 3.11 Melt curves for RPS19 and HPRT1 RT-qPCR assays. Legend: RED - no template control, GRAY - –RT sample, BLUE tumour cDNA, LIGHT GREEN - example of mispriming product, GREEN – spleen cDNA.

57 Chapter 3: Results

Figure 3.12 PD-1, PD-L1 and reference gene RT-qPCR products on agarose gels. Tracks are as follows: 1. RPS19 –RT, 2. RPS19 tumour, 3. RPS19 no template control (NTC), 4. RPS19 positive control (tumour), 5. RPS5 –RT, 6. RPS5 NTC, 7. RPS5 positive control (tumour), 8. PD-L1 –RT, 9. PD-L1 tumour, 10. PD-L1 NTC, 11. PD-L1 positive control (tumour), 12. PD-1 –RT, 13. PD-1 NTC, 14. PD-1 positive control (tumour), 15. HPRT1 –RT, 16. HPRT1 tumour, 17. HPRT1 NTC, 18. HPRT1 positive control (spleen), 19. GUSB –RT, 20. GUSB NTC, 21. GUSB positive control (tumour). Predicted band size for PCR products were as follows: RPS19 95 bp; RPS5 141 bp; PD-L1 186 bp; PD-1 196 bp; HPRT1 114bp; GUSB 103 bp. Positive controls are purified cDNA sequenced PCR product. Arrows indicate examples of primer dimer and mispiriming products.

58 Chapter 3: Results

4.3 Immunohistochemistry for PD-L1

Following validation of a cross-reactive antibody, 26 FFPE samples were stained for PD- L1. All samples were positive for PD-L1 and most of them showed mild to moderate intensity of staining; although 3 samples exhibited strong positivity (case number 1, 14 and 20). Positivity was found both on membrane and cytoplasm. The average score found was 3/5, and 5 tumours had over 81% of cells expressing PD-L1 (Figure 3.13, Table 3.1).

B C

Figure 3.13 PD-L1 staining on FFPE tissue. 40x magnification (50 µm scale bar) A isotype, B positive control on spleen, C tumour.

59 Chapter 4: Discussion

Discussion

The flatcoated retriever (FCR) is one of the dog breeds at risk of developing histiocytic sarcoma (HS) (Fidel et al., 2006) and although the precise cellular origin of this tumour is unknown, the immunophenotype is suggestive of a myeloid dendritic antigen presenting cell (APC) lineage (Kennedy et al., 2016). It has previously been shown that HS in FCR contain a prominent infiltrate of T cells, and

recently that these were regulatory T cells (Treg) phenotypically (Marcinowska et al., 2017). It is interesting that this prominent T cell infiltrate should be present in a tumour comprising cells that modulate the immune response, dendritic cells. It is known that dendritic cells play a pivotal role in determining immune-tolerance versus immunity (Maldonado and Andrian, 2010). Thus a key step in better understanding the relationship between the tumour, its microenvironment and the immune system is essential to understanding how the tumour influences immune function. The finding that a significant proportion of tumour infiltrating T cells

expressed FoxP3, suggesting them to be Treg, raises interesting questions of cause and effect.

The purpose of the present study was to evaluate the relationship between tumour, microenvironment and immune system in canine HS, using flow cytometry, immunohistochemistry and mRNA expression analysis. Specifically, to examine T cell profiles in the peripheral blood of dogs bearing HS by flow cytometry, to document the changes in these profiles, to explore the relationship between tumour

and Treg and the role of the latter in promoting immune-tolerance within the tumour milieu and to explore the expression of tolerogenic immune mediators in HS.

Immunoediting is the process by which the phenotype of neoplastic cells changes under selective pressure; the cells evolve to escape the control of the immune system. This can happen through selection of neoplastic cells that have reduced expression of a particular antigen, but many other mechanisms are involved. Tumour cells can secrete soluble suppressive factors, eg IL-10; they may express immune suppressive molecules, such as PD-L1, on their membrane; and they may

also recruit immunomodulatory cell types such as Treg to the tumour microenvironment (Killick et al., 2015; Naidoo et al., 2015). It was the aim of this project to better investigate these mechanisms in canine HS.

Chapter 4: Discussion

This study found a constant high expression of MHC class II on all samples, no staining for E-cadherin, a percentage of infiltrating lymphocytes stained positive for CD3 and for FoxP3. In almost all samples (25/27) FoxP3 positivity was found on between 1 to 20% of cells. Expression of PD-L1 was identified through immunofluorescence on 3 tumours assessed, however staining varied between tumours. mRNA analysis of PD-L1 expression confirmed expression of the gene in tumour samples although quantitative analysis was not achieved. PD-L1 assessment through immunohistochemistry on archive samples showed an average of 41%- 60% of neoplastic cells staining positive for this marker. The assessment of

peripheral blood through flow cytometry showed no differences in the Treg population between the healthy control group and the dogs bearing HS.

Previous studies have identified canine HS as a tumour of DC. Diagnosis through immunohistochemistry on FFPE samples has been supported (Kato et al., 2013; Moore, 2014). HS is described as a tumour expressing CD18 and MHC class II. Low CD3 and CD79a staining is also expected, and this aids in differentiation from

other round cell tumours of lymphoid origin. Infiltration of Treg within the microenvironment of HS in FCR was demonstrated in a study where FoxP3 positivity was investigated on FFPE tumour samples (Marcinowska et al., 2017). Expression of PD-L1 in canine malignant tumours has been reported in recent literature, however no expression had been identified though immunohistochemistry in HS (Maekawa et al., 2016).

The characteristics of the patient group from which FFPE tissues were used in this study was consistent with that described in the literature (Fidel et al., 2006). The median age at presentation was 8.2 years and there was no sex predisposition.

The samples chosen for the retrospective aspect of this study were from localised tumours, the majority from periarticular lesions. This was to avoid the confusion that may arise when assessing the local cell population within splenic samples. In previous studies, the localised tumours have been shown to have higher numbers of

infiltrating Treg compared to the visceral form (Marcinowska et al., 2017). Localised

Chapter 4: Discussion

tumours are known to have better prognosis compared to visceral ones. This appears

to be in contrast with the hypothesis that Treg down regulate the immune response, producing a worsening of prognosis. However, the severity of the clinical signs typical of the visceral form might be the cause of this apparent discrepancy. Anaemia and intolerance to exercise are less compatible with a good quality of life compared to lameness that is more easily manageable.

Twenty five of the 27 cases selected expressed FoxP3 in cells morphologically identified as lymphocytes. These samples all scored one, meaning that between

1 % and 20 % of the cells were identified as Treg. This agrees with previous finding were 19 % of cells per field, in canine HS, were found positive for FoxP3 (Marcinowska et al., 2017). Although the number of studies is limited, the presence

of infiltrating Treg seems to be correlated with poor prognosis in canine neoplasia, which would be in agreement with the outcome of HS cases (Kim et al., 2012; Sakai

et al., 2018). The prognostic value of increased presence and activity of Treg in human oncology remains controversial (Whiteside, 2015). A recent review identified an overall significant negative effect on survival when assessing the effect of these cells in over 17 types of cancer. However, improved survival correlated to

Treg infiltration of the tumour microenvironment was found in colorectal, head and neck, and oesophageal cancers. It is hypothesised that this might be due to a decreased inflammatory reaction at the tumour site, preventing tissue damage (Salama et al., 2009; Shang et al., 2015; Whiteside, 2015).

Confirmation of diagnosis of HS was achieved through positive staining for CD18 and MHC class II. High expression of MHC class II together with the lack of expression of E-cadherin, identified in this study, is suggestive of a neoplastic population of DC. Possible proliferation of Langherans cells is made unlikely due to the lack of E-cadherin expression. CD79a positivity was not assessed in this study. A recent study conducted in this department had shown no staining for CD79a in canine HS samples (Marcinowska et al., 2017). CD3 intensity of staining was strong in infiltrating lymphocytes, which confirms what was found in the previous project. The consistent presence, identified in this study, of cells

expressing FoxP3 infiltrating the neoplastic cell population also confirms Treg involvement, as reported in previous literature (Marcinowska et al., 2017).

Chapter 4: Discussion

Mature DC are usually regarded as fully differentiated and are not expected to proliferate. Moreover, mature DC are expected to be immunogenic, rather than tolerogenic although this difference is not as rigid (Maldonado and Andrian, 2010). However, natural tolerogenic DC have been extensively studied in specific environments such as intestine and lung (Coombes et al., 2007; Lambrecht and Hammad, 2009; Ostroukhova et al., 2004; Zhang et al., 2001). These cells are important for balancing immune responses to pathogens with those to the regular microbial flora and harmless antigens that are inhaled or swallowed. DC from the + intestinal mucosa and lamina propria have the ability to induce FoxP3 Treg. This is probably due to the mucosal environment rich in anti-inflammatory factors such as TGFβ and IL-10. In skin vitamin D3 appears to play a major role in influencing tolerogenic function in DC (Maldonado and Andrian, 2010). Thus it is possible that the HS tumour cells, even though they are of DC origin, are down regulating any immune response against them by a variety of mechanisms.

In order to determine the stimulus for Treg infiltration of the tumour or induction within the tumour a number of immune mediators were evaluated through immunolabelling of FFPE tumour sections. The PD-1/PD-L1 pathway was investigated. Having failed to achieve convincing staining for PD-1 on FFPE samples, we concentrated our focus on PD-L1 expression. When the immunomodulatory receptor (PD-1) binds with its ligand negative signals are transmitted into the T cell resulting in suppression of antigen-specific immune responses by lymphocytes. This pathway is able to induce an “exhausted” status in antigen-specific lymphocytes aiding immune evasion by tumour cells (Maekawa et al., 2014). Expression of PD-L1 was found in all samples analysed. The average number of neoplastic cells positive for PD-L1 among all tumours was between 41 and 60%. Suggesting that the PD-1/PD-L1 immune checkpoint plays a role in the ability of the tumour to evade the immune system.

The high expression of PD-L1 found on archived samples contrasts with what was found in a study conducted on canine malignant cancers in 2016. This research had assessed the expression of PD-L1, through immunohistochemistry, on 110 samples

Chapter 4: Discussion

of canine malignancies. Samples from oral melanoma, osteosarcoma, hemangiosarcoma, mast cell tumour, mammary and prostate adenocarcinoma had shown positive staining. However, no staining was identified in tissue from canine HS (Maekawa et al., 2016). The breed from which the samples were collected is not specified, nonetheless it is unlikely that the breed would have an influence on the tumour microenvironment if correctly identified as HS. It should be noted that a different antibody was used in this study.

In this project, PD-L1 expression was also high on fresh frozen samples and confirmed by RT-PCR analysis. It was not possible to perform quantitation on the RT-PCR results due to background noise, thus assays would need optimising for future quantitation. Minor adjustments in temperature might improve signal quality (eg. increasing annealing temperature to increase binding specificity). Alternatively, correction or redesign of primers or use of a dual labelled probe based assays might be considered to increase assay specificity. However, the RT-PCR findings remain of great value, as they confirm expression of the PD-L1 gene on the tissue of interest.

Treatment with anti-PD-1 and anti-PD-L1 monoclonal antibodies has recently been approved for a number of tumour types in human oncology (metastatic melanoma, squamous non-small-cell lung carcinoma, renal cell carcinoma, urothelial carcinoma, Hodgkin’s lymphoma, hepatocellular carcinoma, head and neck carcinoma and colorectal cancer) (Naidoo et al., 2015). This therapeutic approach was able to induce durable tumour regression and prolonged stabilisation of the disease (Brahmer et al., 2012). The efficacy of treatments targeting the PD-1/PD- L1 immune check-point may rely on the consequent increased T cell-specific immune response, allowing activation of the immune system to fight the tumour. (Naidoo et al., 2015). Recent studies have shown the capacity of the anti-PD-L1 antibody to reactivate canine anti-tumour immunity in vitro (Maekawa et al., 2014). Novel therapeutic approaches are very much needed for canine HS and the hope is that this project may have indicated a role for this pathway in immune evasion by this cancer and lead to future clinical trials.

Chapter 4: Discussion

The expression of IDO in neoplastic cells may also contribute to the mechanism by + which cells are able to convert T cells into FoxP3 Treg (Munn, 2011). Accumulation of immunosuppressive tryptophan metabolites in tumour cells has been identified as a critical factor in immune evasion. These metabolites have the ability to induce

proliferation of Treg and apoptosis of effective T cells. In human oncology IDO expression has been correlated to poor prognosis in ovarian, endometrial and colorectal cancer (Ino et al., 2008; Mbongue et al., 2015). Again, a cross-reactive antibody that could stain canine IDO in FFPE tissue was not found. However, the optimization of the staining will be resumed for future projects.

A different hypothesis may be that hypoxia within the tumour microenvironment

may be the cause of the increase in Treg. A recent study conducted on canine tumour and lymph node tissue demonstrated a correlation between hypoxia within the

tumour microenvironment and the presence of Treg (Fortuna et al., 2016). Because of the rapid growth of these tumours, it would be reasonable to conjecture that the blood supply would not match the growth, creating areas in which the low oxygen tension would compromise biological functions. This may affect the activity and survival of immune cells. Reports have demonstrated that tumour-infiltrating macrophages display an altered pro-tumour phenotype in hypoxic areas. The transcription factor hypoxia-inducible factor-1 (HIF-1) is suggested to be the central mediator of this hypoxic adaptation (Muraille et al., 2014). Evidence has been found that HS shows higher immunoreactivity to glucose transporter 1 (Glut-1) suggesting that hypoxia plays an important role within these tumours (Abbondati et al., 2013).

It is particularly interesting to investigate induced Treg in HS because of the cell of origin of this tumour (DC). As well as the possible mechanisms discussed above,

HS cells may induce Treg by presentation of modest levels of antigen, in the absence of second signal eg CD80 or relevant cytokines eg IL-12 or by producing anti- inflammatory molecules that modify the environmental conditions and influence T cell differentiation eg TGFβ (Maldonado and Andrian, 2010).

In this study positivity for IL-10 was assessed within the tumour microenvironment.

IL-10 is an important mediator secreted from Treg cells which controls effector T

Chapter 4: Discussion

cell activity. The low level of staining detected suggests that in canine HS IL-10 plays a minor role in induced tumour immune evasion. Only 4 tumours showed detectable expression of IL-10, and in all these it was very low in intensity. This may be a consequence of the difficulty in staining interlukins, as these are secreted and, as a result, liable. However, this may suggest that other cytokines are being

expressed, such as TGFβ or IL-35, also common for Treg activity, or that cell-cell contact is an important mechanism of down-regulation of immune T cells that infiltrate the tumour. A second alternative is that the FoxP3+ T cell phenotype detected is indicative of lack of effector function, eg. IFNγ release, induced by interaction of the infiltrating T cells with the tumour cells rather than induction of

true Treg cells.

Although blood samples were only available from 8 cases of confirmed HS for flow

cytometry analysis in this project, no difference was found in the percentage of Treg in the affected and the healthy control group. This finding suggests that the tumour

cells induced either local differentiation, proliferation or selective migration of Treg to tumour-infiltrated sites (Tominaga et al., 2010). The influence of the microenvironment could be inducing changes on the phenotype of the T cell population, the expression of FoxP3 being induced by cytokines or cell surface ligands within the tumour. If the FoxP3 phenotype is not stable outside the microenvironment, this might be lost when leaving the tissue. Alternatively, it may

suggest that these Treg do not leave the tumour site but die within it, so cannot be identified within the peripheral blood. Because the staining is strong this would suggest that the transcription factor is stably expressed. So continued expression of FoxP3 even when leaving the microenvironment would be expected.

In previous veterinary studies increased levels of Treg have been observed in the peripheral blood of dogs with cancer. Studies have been conducted on dogs with melanoma, osteosarcoma, mammary gland adenocarcinoma, and lymphoma (Biller et al., 2010; Marcinowska et al., 2017; O’Neill et al., 2009; Tominaga et al., 2010). + Most of these studies defined Treg by the expression of FoxP3 in CD4 T cells ie they did not use the CD25 marker. When comparing the level of FoxP3+ CD4+ T

Chapter 4: Discussion

cells in the animals in my study, they were comparable to those in the literature, 4.6% in healthy animals and 6.8% in affected dogs (Biller et al., 2007).

In human oncology an increase in peripheral Treg has been identified in certain types of cancer (Ormandy et al., 2005; Tokuno et al., 2009; Wolf et al., 2003). Interestingly, a decrease was observed following surgical resection of the tumour in patients with gastrointestinal cancer, suggesting that the neoplastic cells were

involved in the expansion of the Treg pool (Tokuno et al., 2009).

Another factor that should be taken into account is that the level of C reactive protein in the blood of dogs with HS is increased (Tagawa et al., 2016). These dogs might be mounting an inflammatory response to the tumour, through either necrosis or cytokine production, or both. This opposing effect on lymphocyte populations

may explain the lack of increased Treg percentage peripherally.

+ + CD4 CD25 Treg are a population of suppressor T cells that play an important role in maintaining peripheral immune tolerance (Sakaguchi, 2000). Although the vast majority of these cells originate from the thymus, there is growing evidence

suggesting that Treg may also be induced in the periphery (Chen et al., 2003). These cells are thought to be derived from CD4+CD25- T cells that are converted to + + + CD4 CD25 FoxP3 Treg following antigen exposure. A CD8/CD25/FoxP3 panel + was designed, to allow investigation of induced CD8 Treg as induced cells can differ in their T cell of origin (Maldonado and Andrian, 2010). However, because of the limited number of cells in the samples, priority was given to the CD4/CD25/FoxP3 panel. One only sample was analysed with the CD8 antibody for

alternative Treg (results not reported), thus was of no statistical value. This probably does not alter the results of this project in comparison to the reviewed literature, as

the majority of the previous publications investigated Treg cells as a population expressing CD4.

An age-matched control group was chosen to account for the changes in the T cell compartment due to age. Studies conducted in humans have demonstrated an

increase in natural Treg in peripheral blood of aged individuals. However, murine

Chapter 4: Discussion

and human models have suggested that older individuals have reduced capacity to

generate iTreg (Jagger et al., 2014).

A further aspect of interest is the Treg/CD8 ratio. In studies conducted in human and

veterinary oncology a correlation has been found between the degree of Treg infiltration within the tumour and suppression of CD8+ T cell effectiveness. A

recent study conducted on different types of cancer found an increased Treg/CD8 T cell ratio within the peripheral blood of dogs with different cancers (O’Neill et al., 2009). No significant difference was found within the samples analysed for this project, despite the reduction in percentage of CD8+ T cells. This reduction in the proportion of CD8+ T cells observed in the tumour group suggests that there was a reduction in cytotoxic T effector cells in affected dogs.

A trend was observed towards a reduction of CD3+ cells in the peripheral blood of dogs diagnosed with HS. This trend in decreased T cell levels was only apparent in the cytotoxic T cells ie CD8+ T cells which showed significantly reduced proportions in the peripheral blood of tumour bearing dogs. However there was a contrasting increase in the percentage of CD4-CD8- T lymphocytes in the peripheral blood of animals bearing HS tumours. It was not possible to identify if these changes are specific to HS or recurrent in different cancers. Analysing samples from dogs bearing different tumours would help elucidate if these changes are restricted to HS only.

This increased percentage of CD3+CD4-CD8- T cells in dogs with HS suggests an involvement of γδ T cells. These T cells are usually not MHC restricted in the same way as  T cells, but recognise (by both their TCR and NK-type receptors) a diverse set of molecules including stress induced molecules, microbial metabolites or eukaryotic isoprenoid precursor, IPP, or lipid antigens presented on CD1. After activation,  T cells produce cytokines including IFN- and IL-17, are cytolytic via perforin and granzymes and TRAIL expression, and can act as antigen presenting cells. Due to metabolic dysregulation IPP often accumulates in cancer cells and  T cells are known to be able to recognise cancer cells in an MHC

Chapter 4: Discussion

unrestricted manner. This suggest that they could be used therapeutically (Abe et al., 2009; Mirzaei et al., 2016). Despite the increased percentage of CD3+CD4-CD8- T cells, animals with HS have poor prognosis and so it is unclear if these cells are functioning in an anti-tumour manner in this disease.

It was of interest to investigate the expression of perforin and granzyme to better understand the activity of CD3+CD8+ T cells. Cytotoxic T cells are central to immune surveillance of cancer. Their ability to form pores through the protein perforin and for this to cause the transfer of granzymes into the cytoplasm of the target cell is what gives them the ability to kill neoplastic cells though apoptosis (Voskoboinik et al., 2015). Unfortunately antibody validation for these proteins on FFPE tissue was unsuccessful. However, the diminished presence of CD3+CD8+ T cells within the peripheral blood may reflect a diminished presence and activity within the microenvironment, partly explaining the poor effectiveness of the immune response to this tumour.

Limitations of the retrospective aspect of this project are due to the use of FFPE archive samples influencing the choice of antibodies available. With fresh or frozen tissue staining for CD1 and CD11c would have been performed to confirm myeloid dendritic antigen presenting cell (APC) origin. Ideally, staining for CD79a should have also been attempted, to definitively exclude cells of lymphoid origin. Double staining would have also been very advantageous to identify multiple markers being expressed by the same cell. A further limitation of this study lies in the subjectivity of scoring slides manually. All the slides stained for this project have been digitized with a slide scanner. Unfortunately machine training requires hundreds of slides to be able to objectively assess positive staining, so this could not have been achieved with the numbers collected for this project. Collecting more samples and expanding the database will allow more objective scoring of the immunolabelling in the future.

During the validation of anti-PD-L1 antibody, immunofluorescence was of great help. However the lack of control canine tissue was an issue, especially because the antibody was not raised against dog-derived antigen. For this project we chose to use margins of the tumour as a control. At the time of writing we are assessing

Chapter 4: Discussion

expression in healthy subcutaneous dog tissue and cross referencing this with the staining pattern in the tumour samples.

The prospective approach of this project also comes with limitations. Based on the number of FCR affected with HS referred to the Queen’s Veterinary Hospital in the previous years we had hoped to collect blood from a minimum of 10 affected dogs. In the year of this study, fewer cases were referred and only 8 affected dogs were available for this study. The necessity to process samples within 24 hours from collection made sample donations from other veterinary clinics sparse. However even with these limitations significant differences were detectable within the T cell populations of the affected dogs. Due to the prospective nature of this aspect of the project we were unable to recover samples from tumour draining lymph nodes to

analyse by flow cytometry. Other researchers have found a greater increase of Treg within the draining lymph nodes than in peripheral blood, although this increase was commonly also reflected in peripheral blood (Biller et al., 2007). Additionally, the flow cytometer used was a four colour machine, the use of more colour markers would allow a fuller investigation of these very small samples. However in the canine system it has proven difficult to find directly conjugated antibodies that could be used.

Because the two parts of this project were conducted on different sample groups, the blood samples and the FFPE samples do not belong to the same patients. Going forward it would be ideal to have tissue and blood from the same animal to be able to correlate microenvironment and peripheral blood. Fresh tumour samples would allow disaggregation and interrogation of the infiltrating T cells by flow cytometry

and for function eg are they truly Treg. Hopefully incrementing the samples will increase the size of both groups and will improve the statistical power of the study.

Chapter 4: Discussion

Conclusions and future work

+ This MPhil study has confirmed the presence of FoxP3 Treg within the tumour microenvironment of canine HS, and the results are suggestive of a localised effect linked to the tumour environment.

Since the results of this project suggest the presence of Treg is linked to the tumour microenvironment, the role played by TGF-β becomes of interest as antigen presentation by dendritic cells in the presence of this cytokine results in

differentiation of Treg. Expansion of Treg can be induced by high concentrations of TGF-β, which may be produced by the tumour itself (Fu et al., 2004).

Future work should also investigate in parallel the C reactive protein concentrations in the HS dog samples as this might help clarify the cause of the lack of change of

Treg in peripheral blood.

This project has identified a possible role for the PD-1/PD-L1 pathway in canine HS immune evasion. This should be investigated further. Promisingly, there has been a recent Japanese patent application (2016-159088) for a canine chimeric monoclonal antibody targeting PD-L1 and its efficacy has been tested in a clinical trial on oral malignant melanoma (1 out of 7 dogs treated showed obvious tumour regression) and undifferentiated sarcoma (1 out of 2 dogs clearly responded to treatment). It is encouraging that none of the dogs in this clinical trial exhibited allergic reactions or autoimmune disease (Maekawa et al., 2017). Clinical trials in dogs bearing HS might be a possibility in the near future.

References

Abadie, J., Hédan, B., Cadieu, E., De Brito, C., Devauchelle, P., Bourgain, C., Parker, H.G., Vaysse, A., Margaritte-Jeannin, P., Galibert, F., Ostrander, E.A., André, C., 2009. Epidemiology, pathology, and genetics of histiocytic sarcoma in the bernese mountain dog breed. Journal of Heredity 100, S19–S27. https://doi.org/10.1093/jhered/esp039

Abbondati, E., Del-Pozo, J., Hoather, T.M., Constantino-Casas, F., Dobson, J.M., 2013. An immunohistochemical study of the expression of the hypoxia markers Glut-1 and Ca-IX in canine sarcomas. Veterinary pathology 50, 1063–9. https://doi.org/10.1177/0300985813486810

Abe, Y., Muto, M., Nieda, M., Nakagawa, Y., Nicol, A., Kaneko, T., Goto, S., Yokokawa, K., Suzuki, K., 2009. Clinical and immunological evaluation of zoledronate-activated Vγ9γδ T-cell-based immunotherapy for patients with multiple myeloma. Experimental Hematology 37, 956–968. https://doi.org/10.1016/j.exphem.2009.04.008

Affolter, V.K., Moore, P.F., 2002. Localized and Disseminated Histiocytic Sarcoma of Dendritic Cell Origin in Dogs. Vet Pathol 39, 74–83.

Alvarez, D., Vollmann, E.H., von Andrian, U.H., 2008. Mechanisms and Consequences of Dendritic Cell Migration. Immunity 29, 325–342. https://doi.org/10.1016/j.immuni.2008.08.006

Angelo, S.P.D., Shoushtari, A.N., Agaram, N.P., Kuk, D., Qin, L., Carvajal, R.D., Dickson, M.A., Gounder, M., Louise, M., Schwartz, G.K., Tap, W.D., 2015. Prevalence of tumor-infiltrating lymphocytes and PD-L1 expression in the soft tissue sarcoma. Human Pathology 46, 357–365. https://doi.org/10.1016/j.humpath.2014.11.001

Apostolou, I., von Boehmer, H., 2004. In Vivo Instruction of Suppressor Commitment in Naive T Cells. The Journal of Experimental Medicine 199, 1401–1408. https://doi.org/10.1084/jem.20040249

Ayers, D., Clements, D.N., Salway, F., Day, P.J.R., 2007. Expression stability of

commonly used reference genes in canine articular connective tissues. BMC Veterinary Research 3, 1–10. https://doi.org/10.1186/1746-6148-3-7

Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y., Pulendran, B., Palucka, K., 2000. Immunobiology of dendritic cells. Annual reviews 767–811.

Beissert, S., Schwarz, A., Schwarz, T., 2006. Regulatory T Cells. Journal of Investigative Dermatology 126, 15–24. https://doi.org/10.1038/sj.jid.5700004

Belkaid, Y., Rouse, B.T., 2005. Natural regulatory T cells in infectious disease. Nature Immunology 6, 353–360. https://doi.org/10.1038/ni1181

Biller, B.J., Elmslie, R.E., Burnett, R.C., Avery, A.C., Dow, S.W., 2007. Use of FoxP3 expression to identify regulatory T cells in healthy dogs and dogs with cancer. Veterinary Immunology and Immunopathology 116, 69–78. https://doi.org/10.1016/j.vetimm.2006.12.002

Biller, B.J., Guth, A., Burton, J.H., Dow, S.W., 2010. Decreased Ratio of CD8+ T Cells to Regulatory T Cells Associated with Decreased Survival in Dogs with Osteosarcoma. Journal of veterinary internal medicine / American College of Veterinary Internal Medicine 24, 1118–1123. https://doi.org/10.1016/j.micinf.2011.07.011.Innate

Boerkamp, K.M., van der Kooij, M., van Steenbeek, F.G., van Wolferen, M.E., Groot Koerkamp, M.J.A., van Leenen, D., Grinwis, G.C.M., Penning, L.C., Wiemer, E.A.C., Rutteman, G.R., 2013. Gene Expression Profiling of Histiocytic Sarcomas in a Canine Model: The Predisposed Flatcoated Retriever Dog. PLoS ONE 8. https://doi.org/10.1371/journal.pone.0071094

Brahmer, J.R., Tykodi, S.S., Chow, L.Q., Hwu, W.J., Topalian, S.L., Hwu, P., Drake, C.G., Camacho, L.H., Kauh, J., Odunsi, K., Pitot, H.C., Hamid, O., Bhatia, S., Martins, R., Eaton, K., Chen, S., Salay, T.M., Alaparthy, S., Grosso, J.F., Korman, A.J., Parker, S.M., Agrawal, S., Goldberg, S.M., Pardoll, D.M., Gupta, A., Wigginton, J.M., 2012. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. New England Journal of Medicine 366, 2455–2465. https://doi.org/10.1056/NEJMoa1200694

Brinkhof, B., Spee, B., Rothuizen, J., Penning, L.C., 2006. Development and evaluation of canine reference genes for accurate quantification of gene expression. Analytical Biochemistry 356, 36–43. https://doi.org/10.1016/j.ab.2006.06.001 73

Bulliard, Y., Jolicoeur, R., Zhang, J., Dranoff, G., Wilson, N.S., Brogdon, J.L., 2014. OX40 engagement depletes intratumoral Tregs via activating FcγRs, leading to antitumor efficacy. Immunology and Cell Biology 92, 475–480. https://doi.org/10.1038/icb.2014.26

Cannon, C., Borgatti, A., Henson, M., Husbands, B., 2015. Evaluation of a combination chemotherapy protocol including lomustine and doxorubicin in canine histiocytic sarcoma. Journal of Small Animal Practice 56, 425–429. https://doi.org/10.1111/jsap.12354

Carvalho, M.I., Pires, I., Prada, J., Gregório, H., Lobo, L., Queiroga, F.L., 2016. Intratumoral FoxP3 expression is associated with angiogenesis and prognosis in malignant canine mammary tumors. Veterinary Immunology and Immunopathology 178, 1–9. https://doi.org/10.1016/j.vetimm.2016.06.006

Carvalho, M.I., Pires, I., Prada, J., Queiroga, F.L., 2014. A role for T-lymphocytes in human breast cancer and in canine mammary tumors. BioMed Research International 2014. https://doi.org/10.1155/2014/130894

Chandra, A.M.S., Ginn, P.E., 1999. Primary malignant histiocytosis of the brain in a dog. Journal of Comparative Pathology 121, 77–82. https://doi.org/10.1053/jcpa.1998.0296

Chen, W., Jin, W., Hardegen, N., Lei, K., Li, L., Marinos, N., McGrady, G., Wahl, S.M., 2003. Conversion of Peripheral CD4 + CD25 − Naive T Cells to CD4 + CD25 + Regulatory T Cells by TGF-β Induction of Transcription Factor Foxp3. The Journal of Experimental Medicine 198, 1875–1886. https://doi.org/10.1084/jem.20030152

Clements, D.N., Carter, S.D., Innes, J.F., Ollier, W.E.R., Day, P.J.R., 2006. Analysis of normal and osteoarthritic canine cartilage mRNA expression by quantitative polymerase chain reaction. Arthritis research & therapy 8, R158. https://doi.org/10.1186/ar2053

Constantino-Casas, F., Mayhew, D., Hoather, T.M., Dobson, J.M., 2011. The Clinical Presentation and Histopathologic–Immunohistochemical Classification of Histiocytic Sarcomas in the Flat Coated Retriever. Veterinary Pathology 48, 764– 771. https://doi.org/10.1177/0300985810385153

Coombes, J.L., Siddiqui, K.R.R., Arancibia-Cárcamo, C. V., Hall, J., Sun, C.-M., Belkaid, Y., Powrie, F., 2007. A functionally specialized population of mucosal 74

CD103 + DCs induces Foxp3 + regulatory T cells via a TGF-β– and retinoic acid– dependent mechanism. The Journal of Experimental Medicine 204, 1757–1764. https://doi.org/10.1084/jem.20070590

Coomer, A.R., Liptak, J.M., 2008. Canine histiocytic diseases. Compendium (Yardley, PA) 30, 202–4, 208-16-17.

Craig, L.E., Julian, M.E., Ferracone, J.D., 2002. The Diagnosis and Prognosis of Synovial Tumors in Dogs: 35 Cases. Veterinary Pathology 39, 66–73. https://doi.org/10.1354/vp.39-1-66

Curti, B.D., Kovacsovics-Bankowski, M., Morris, N., Walker, E., Chisholm, L., Floyd, K., Walker, J., Gonzalez, I., Meeuwsen, T., Fox, B.A., Moudgil, T., Miller, W., Haley, D., Coffey, T., Fisher, B., Delanty-Miller, L., Rymarchyk, N., Kelly, T., Crocenzi, T., Bernstein, E., Sanborn, R., Urba, W.J., Weinberg, A.D., 2013. OX40 is a potent immune-stimulating target in late-stage cancer patients. Cancer Research 73, 7189–7198. https://doi.org/10.1158/0008-5472.CAN-12-4174

D’Agostino, P.M., Gottfried-Blackmore, A., Anandasabapathy, N., Bulloch, K., 2012. Brain dendritic cells: Biology and pathology. Acta Neuropathologica 124, 599–614. https://doi.org/10.1007/s00401-012-1018-0

Dobson, J., Villiers, E., Roulois, A., Gould, S., Mellor, P., Hoather, T., Watson, P., 2006. Histiocytic sarcoma of the spleen in flat-coated retrievers with regenerative anaemia and hypoproteinaemia. Veterinary Records 158, 825–829. https://doi.org/10.1136/vr.158.24.825

Dobson, J.M., 2013. Breed-predispositions to cancer in pedigree dogs. ISRN veterinary science 2013, 941275. https://doi.org/10.1155/2013/941275

Duraiswamy, J., Freeman, G.J., Coukos, G., 2013. Therapeutic PD-1 Pathway Blockade Augments with Other Modalities of Immunotherapy T-Cell Function to Prevent Immune Decline in Ovarian Cancer. Cancer Research 73(23):6900-12. https://doi.org/10.1158/0008-5472.CAN-13-1550

Erich, S.A., Rutteman, G.R., Teske, E., 2013. Causes of death and the impact of histiocytic sarcoma on the life expectancy of the Dutch population of Bernese mountain dogs and Flat-coated retrievers. Veterinary Journal 198, 678–683. https://doi.org/10.1016/j.tvjl.2013.09.062

Fant, P., Caldin, M., Furlanello, T., De Lorenzi, D., Bertolini, G., Bettini, G., Morini, M.,

Masserdotti, C., 2004. Primary gastric histiocytic sarcoma in a dog--a case report. Journal of veterinary medicine Series A 51(7-8):358-62. https://doi.org/10.1111/j.1439-0442.2004.00645.x

Fidel, J., Schiller, I., Hauser, B., Jausi, Y., Rohrer-Bley, C., Roos, M., Kaser-Hotz, B., 2006. Histiocytic sarcomas in flat-coated retrievers: a summary of 37 cases (November 1998-March 2005). Veterinary and comparative oncology 4, 63–74. https://doi.org/DOI 10.1111/j.1476-5810.2006.00090.x

Fontenot, J.D., Gavin, M.A., Rudensky, A.Y., 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nature Immunology 4, 330–336. https://doi.org/10.1038/ni904

Fortuna, L., Relf, J., Chang, Y.M., Hibbert, A., Martineau, H.M., Garden, O.A., 2016. Prevalence of FoxP3+Cells in Canine Tumours and Lymph Nodes Correlates Positively with Glucose Transporter 1 Expression. Journal of Comparative Pathology 155, 171–180. https://doi.org/10.1016/j.jcpa.2016.06.006

Francisco, L., Salinas, V., Brown, K., Vanguri, V., Freeman, G., Kuchroo, V., Sharpe, A., 2009. OR.100. PD-L1 Regulates the Development, Maintenance and Function of Induced-regulatory T Cells. Clinical Immunology 131, S41. https://doi.org/10.1016/j.clim.2009.03.115

Freeman, B.G.J., Long, A.J., Iwai, Y., Bourque, K., Chernova, T., Nishimura, H., Fitz, L.J., Malenkovich, N., Okazaki, T., Byrne, M.C., Horton, H.F., Fouser, L., Carter, L., Ling, V., Bowman, M.R., Carreno, B.M., Collins, M., Wood, C.R., Honjo, T., 2000. Engagement of the PD-1 Immunoinhibitory Receptor by a Novel B7 Family Member Leads to Negative Regulation of Lymphocyte Activation. The Journal of Experimental Medicine 192(7):1027-34.

Fu, S., Zhang, N., Yopp, A.C., Chen, D., Mao, M., Chen, D., Zhang, H., Ding, Y., Bromberg, J.S., 2004. TGF-β induces Foxp3 + T-regulatory cells from CD4 + CD25 - precursors. American Journal of Transplantation 4, 1614–1627. https://doi.org/10.1111/j.1600-6143.2004.00566.x

Fulmer, A.K., Mauldin, G.E., 2007. Canine histiocytic neoplasia: An overview. Canadian Veterinary Journal 48, 1041–1050. https://doi.org/10.1515/semi.1969.1.3.339

Garden, O.A., Pinheiro, D., Cunningham, F., 2011. All creatures great and small: Regulatory T cells in mice, humans, dogs and other domestic animal species.

International Immunopharmacology 11(5):576-88. https://doi.org/10.1016/j.intimp.2010.11.003

H. Munn, D., 2011. Indoleamine 2,3-dioxygenase, Tregs and Cancer. Current Medicinal Chemistry 18, 2240–2246. https://doi.org/10.2174/092986711795656045

Hafeman, S., London, C., Elmslie, R., Dow, S., 2010. Evaluation of liposomal clodronate for treatment of malignant histiocytosis in dogs. Cancer Immunology, Immunotherapy 59, 441–452. https://doi.org/10.1007/s00262-009-0763-y

Hafeman, S.D., Varland, D., Dow, S.W., 2012. Bisphosphonates significantly increase the activity of doxorubicin or vincristine against canine malignant histiocytosis cells. Veterinary and Comparative Oncology 10, 44–56. https://doi.org/10.1111/j.1476- 5829.2011.00274.x

Hartley, G., Faulhaber, E., Caldwell, A., Coy, J., Kurihara, J., Guth, A., Regan, D., Dow, S., 2016. Immune regulation of canine tumour and macrophage PD-L1 expression. Veterinary and Comparative Oncology 15 (2) 534–549. https://doi.org/10.1111/VCO.12197

Hatziioannou, A., Alissafi, T., Verginis, P., 2017. Myeloid-derived suppressor cells and T regulatory cells in tumors: unraveling the dark side of the force. Journal of Leukocyte Biology. https://doi.org/10.1189/jlb.5VMR1116-493R

Ichihara, F., Kono, K., Takahashi, A., Kawaida, H., Sugai, H., Fujii, H., 2003. Increased populations of regulatory T cells in peripheral blood and tumor-infiltrating lymphocytes in patients with gastric and esophageal cancers. Clinical cancer research : an official journal of the American Association for Cancer Research 9, 4404–4408.

Ino, K., Yamamoto, E., Shibata, K., Kajiyama, H., Yoshida, N., Terauchi, M., Nawa, A., Nagasaka, T., Takikawa, O., Kikkawa, F., 2008. Inverse correlation between tumoral indoleamine 2,3-dioxygenase expression and tumor-infiltrating lymphocytes in endometrial cancer: Its association with disease progression and survival. Clinical Cancer Research 14, 2310–2317. https://doi.org/10.1158/1078-0432.CCR-07-4144

Ito, K., Kuroki, S., Kobayashi, M., Ono, K., Washizu, T., Bonkobara, M., 2013. Identification of dasatinib as an in vitro potent growth inhibitor of canine histiocytic sarcoma cells. Veterinary Journal. https://doi.org/10.1016/j.tvjl.2012.12.016

Jagger, A., Shimojima, Y., Goronzy, J.J., Weyand, C.M., 2014. Regulatory T cells and

the immune aging process: A mini-review. Gerontology 60, 130–137. https://doi.org/10.1159/000355303

Kaim, U., Moritz, A., Failing, K., Baumgärtner, W., 2006. The regression of a canine Langerhans cell tumour is associated with increased expression of IL-2, TNF-α, IFN-γ and iNOS mRNA. Immunology 118, 472–482. https://doi.org/10.1111/j.1365- 2567.2006.02394.x

Kato, Y., Murakami, M., Hoshino, Y., Mori, T., Maruo, K., Hirata, A., Nakagawa, T.L.D.R., Yanai, T., Sakai, H., 2013. The Class A Macrophage Scavenger Receptor CD204 is a Useful Immunohistochemical Marker of Canine Histiocytic Sarcoma. Journal of Comparative Pathology 148, 188–196. https://doi.org/10.1016/j.jcpa.2012.06.009

Kennedy, K., Thomas, R., Breen, M., 2016. Canine Histiocytic Malignancies— Challenges and Opportunities. Veterinary Sciences 3, 2. https://doi.org/10.3390/vetsci3010002

Killick, D.R., Stell, A.J., Catchpole, B., 2015. Immunotherapy for canine cancer - Is it time to go back to the future? Journal of Small Animal Practice 56, 229–241. https://doi.org/10.1111/jsap.12336

Kim, J.H., Chon, S.K., Im, K.S., Kim, N.H., Cho, K.W., Sur, J.H., 2013. Infiltrating Foxp3+ Regulatory T cells and histopathological features in canine classical and spermatocytic seminomas. Reproduction in Domestic Animals 48, 218–222. https://doi.org/10.1111/j.1439-0531.2012.02135.x

Kim, J.H., Hur, J.H., Lee, S.M., Im, K.S., Kim, N.H., Sur, J.H., 2012. Correlation of Foxp3 positive regulatory T cells with prognostic factors in canine mammary carcinomas. Veterinary Journal 193, 222–227. https://doi.org/10.1016/j.tvjl.2011.10.022

Knueppel, A., Lange, S., Sekora, A., Altmann, S., Freund, M., Junghanss, C., 2011. Phenotypic and Functional Characterization of Freshly Isolated and Expanded Canine Regulatory T Cells. Experimental Animals 60, 471–479.

Lambrecht, B.N., Hammad, H., 2009. Biology of Lung Dendritic Cells at the Origin of Asthma. Immunity 31, 412–424. https://doi.org/10.1016/j.immuni.2009.08.008

Lio, C.-W.J., Hsieh, C.-S., 2008. A two-step process for thymic regulatory T cell development. Immunity 28, 100–11. https://doi.org/10.1016/j.immuni.2007.11.021

Liu, S., Liu, D., Li, J., Zhang, D., Chen, Q., 2016. Regulatory T cells in oral squamous cell carcinoma. Journal of Oral Pathology & Medicine 45, 635–639. https://doi.org/10.1111/jop.12445

Liyanage, U.K., Moore, T.T., Joo, H.-G., Tanaka, Y., Herrmann, V., Doherty, G., Drebin, J.A., Strasberg, S.M., Eberlein, T.J., Goedegebuure, P.S., Linehan, D.C., 2002. Prevalence of Regulatory T Cells Is Increased in Peripheral Blood and Tumor Microenvironment of Patients with Pancreas or Breast Adenocarcinoma. The Journal of Immunology 169, 2756–2761. https://doi.org/10.4049/jimmunol.169.5.2756

Looringh Van Beeck, F.A., Zajonc, D.M., Moore, P.F., Schlotter, Y.M., Broere, F., Rutten, V.P.M.G., Willemse, T., Van Rhijn, I., 2008. Two canine CD1a proteins are differentially expressed in skin. Immunogenetics 60, 315–324. https://doi.org/10.1007/s00251-008-0297-z

Maeda, S., Ohno, K., Fujiwara-Igarashi, A., Uchida, K., Tsujimoto, H., 2016. Changes in Foxp3-Positive Regulatory T Cell Number in the Intestine of Dogs With Idiopathic Inflammatory Bowel Disease and Intestinal Lymphoma. Veterinary Pathology 53, 102–112. https://doi.org/10.1177/0300985815591081

Maekawa, N., Konnai, S., Ikebuchi, R., Okagawa, T., Adachi, M., Takagi, S., Kagawa, Y., Nakajima, C., Suzuki, Y., Murata, S., Ohashi, K., 2014. Expression of PD-L1 on canine tumor cells and enhancement of IFN-γ production from tumor-infiltrating cells by PD-L1 blockade. PLoS ONE 9. https://doi.org/10.1371/journal.pone.0098415

Maekawa, N., Konnai, S., Okagawa, T., Nishimori, A., Ikebuchi, R., Izumi, Y., Takagi, S., Kagawa, Y., Nakajima, C., Suzuki, Y., Kato, Y., Murata, S., Ohashi, K., 2016. Immunohistochemical analysis of PD-L1 expression in canine malignant cancers and PD-1 expression on lymphocytes in canine oral melanoma. PLoS ONE 11, 1–13. https://doi.org/10.1371/journal.pone.0157176

Maekawa, N., Konnai, S., Takagi, S., Kagawa, Y., Okagawa, T., Nishimori, A., Ikebuchi, R., Izumi, Y., Deguchi, T., Nakajima, C., Kato, Y., Yamamoto, K., Uemura, H., Suzuki, Y., Murata, S., Ohashi, K., 2017. A canine chimeric monoclonal antibody targeting PD-L1 and its clinical efficacy in canine oral malignant melanoma or undifferentiated sarcoma. Scientific Reports 7, 1–12. https://doi.org/10.1038/s41598- 017-09444-2

Maldonado, R., Andrian, U. Von, 2010. How tolerogenic dendritic cells induce regulatory T cells, Advances in immunology. https://doi.org/10.1016/B978-0-12-380995- 7.00004-5.How

Marcinowska, A., Constantino-Casas, F., Williams, T., Hoather, T., Blacklaws, B., Dobson, J., 2017. T Lymphocytes in Histiocytic Sarcomas of Flat-Coated Retriever Dogs. Veterinary Pathology 30098581769020. https://doi.org/10.1177/0300985817690208

Mariani, C.L., Jennings, M.K., Olby, N.J., Borst, L.B., Brown, J.C., Robertson, I.D., Seiler, G.S., Mackillop, E., 2015. Histiocytic Sarcoma with Central Nervous System Involvement in Dogs: 19 Cases (2006-2012). Journal of Veterinary Internal Medicine 29, 607–613. https://doi.org/10.1111/jvim.12554

Mazzone, A., Ricevuti, G., 1995. Leukocyte CD11/CD18 integrins: Biological and clinical relevance. Haematologica 80, 161–175.

Mbongue, J., Nicholas, D., Torrez, T., Kim, N.-S., Firek, A., Langridge, W., 2015. The Role of Indoleamine 2, 3-Dioxygenase in Immune Suppression and Autoimmunity. Vaccines 3, 703–729. https://doi.org/10.3390/vaccines3030703

Merad, M., Ginhoux, F., Collin, M., 2008. Origin, homeostasis and function of Langerhans cells and other langerin-expressing dendritic cells. Nature reviews. Immunology 8, 935–47. https://doi.org/10.1038/nri2455

Mirzaei, H.R., Mirzaei, H., Yun Lee, S., Hadjati, J., Till, B.G., 2016. Prospects for chimeric antigen receptor (CAR) γδ T cells: A potential game changer for adoptive T cell cancer immunotherapy. Cancer Letters 380, 413–423. https://doi.org/10.1016/j.canlet.2016.07.001

Moore, A., Taylor, D., Reppas, G., Frimberger, A., 2017. Chemotherapy for dogs with lymph node metastasis from histiocytic sarcomas. Australian Veterinary Journal. https://doi.org/10.1111/avj.12522

Moore, P.F., 2014. A Review of Histiocytic Diseases of Dogs and Cats. Veterinary Pathology 51, 167–184. https://doi.org/10.1177/0300985813510413

Moore, P.F., Affolter, V.K., Vernau, W., 2006. Canine hemophagocytic histiocytic sarcoma: a proliferative disorder of CD11d+ macrophages. Veterinary Pathology 43, 632–645. https://doi.org/10.1354/vp.43-5-632

Moore, P.F., Schrenzel, M.D., Affolter, V.K., Olivry, T., Naydan, D., 1996. Canine 80

cutaneous histiocytoma is an epidermotropic Langerhans cell histiocytosis that expresses CD1 and specific beta 2-integrin molecules. The American journal of pathology 148, 1699–708.

Morris, J.S., McInnes, E.F., Bostock, D.E., Hoather, T.M., Dobson, J.M., 2002. Immunohistochemical and histopathologic features of 14 malignant fibrous histiocytomas from Flat-Coated Retrievers. Veterinary pathology 39, 473–479. https://doi.org/10.1354/vp.39-4-473

Muenst, S., Schaerli, A.R., Gao, F., Daster, S., Trella, E., Droeser, R.A., Muraro, M.G., Zajac, P., Zanetti, R., Gillanders, W.E., Weber, W.P., Soysal, S.D., 2014. Expression of programmed death ligand 1 ( PD-L1 ) is associated with poor prognosis in human breast cancer. Breast cancer research and treatment 146(1), 15–24. https://doi.org/10.1007/s10549-014-2988-5

Munn, H. D., 2011. Indoleamine 2,3-dioxygenase, Tregs and Cancer. Current Medicinal Chemistry 18, 2240–2246. https://doi.org/10.2174/092986711795656045

Muraille, E., Leo, O., Moser, M., 2014. Th1/Th2 paradigm extended: Macrophage polarization as an unappreciated pathogen-driven escape mechanism? Frontiers in Immunology 5, 1–12. https://doi.org/10.3389/fimmu.2014.00603

Naidoo, J., Page, D.B., Li, B.T., Connell, L.C., Schindler, K., Lacouture, M.E., Postow, M.A., Wolchok, J.D., 2015. Toxicities of the anti-PD-1 and anti-PD-L1 immune checkpoint antibodies. Annals of Oncology 26, 2375–2391. https://doi.org/10.1093/annonc/mdv383

O’Neill, K., Guth, A., Biller, B., Elmslie, R., Dow, S., 2009. Changes in regulatory T cells in dogs with cancer and associations with tumor type. Journal of Veterinary Internal Medicine 23, 875–881. https://doi.org/10.1111/j.1939-1676.2009.0333.x

Ormandy, L.A., Hillemann, T., Wedemeyer, H., Manns, M.P., Greten, T.F., Korangy, F., 2005. Increased Populations of Regulatory T Cells in Peripheral Blood of Patients with Hepatocellular Carcinoma. Cancer Research 65, 2457–2464. https://doi.org/10.1158/0008-5472.CAN-04-3232

Ostroukhova, M., Seguin-devaux, C., Oriss, T.B., Dixon-mccarthy, B., Yang, L., Ameredes, B.T., Corcoran, T.E., Ray, A., 2004. Tolerance induced by inhaled antigen involves CD4 + T cells expressing membrane bound TGFβ and FoxP3. The journal of clinical investigation 114(1):28-38.

https://doi.org/10.1172/JCI200420509.28

Pierezan, F., Mansell, J., Ambrus, A., Hoffmann, A.R., 2014. Immunohistochemical expression of ionized calcium binding adapter molecule 1 in cutaneous histiocytic proliferative, neoplastic and inflammatory disorders of dogs and cats. Journal of Comparative Pathology 151, 347–351. https://doi.org/10.1016/j.jcpa.2014.07.003

Pinheiro, D., Singh, Y., Grant, C.R., Appleton, R.C., Sacchini, F., Walker, K.R.L., Chadbourne, A.H., Palmer, C.A., Armitage-Chan, E., Thompson, I., Williamson, L., Cunningham, F., Garden, O.A., 2011. Phenotypic and functional characterization of a CD4+ CD25high FOXP3high regulatory T-cell population in the dog. Immunology 132, 111–122. https://doi.org/10.1111/j.1365-2567.2010.03346.x

Pires, I., Rodrigues, P., Alves, A., Queiroga, F.L., Silva, F., Lopes, C., 2013. Immunohistochemical and immunoelectron study of major histocompatibility complex class-II antigen in canine cutaneous histiocytoma: Its relation to tumor regression. In Vivo 27(2):257-62.

Ramirez, S., Douglass, P., Robertson, D., 2005. Ultrasonographic features of canine abdominal malignant histiocytosis. Veterinary Radiology and Ultrasound 43(2), 167–170.

Rissetto, K.C., Rindt, H., Selting, K.A., Villamil, J.A., Henry, C.J., Reinero, C.R., 2010. Cloning and expression of canine CD25 for validation of an anti-human CD25 antibody to compare T regulatory lymphocytes in healthy dogs and dogs with osteosarcoma. Veterinary Immunology and Immunopathology 135, 137–145. https://doi.org/10.1016/j.vetimm.2010.02.002

Rosin, A., 1986. Malignant Histiocytosis of Bernese Mountain Dogs. Journal of the American Veterinary Medicine Association 10, 1–10.

Rossi, S., Gelain, M.E., Comazzi, S., 2009. Disseminated histiocytic sarcoma with peripheral blood involvement in a Bernese Mountain dog. Veterinary Clinical Pathology. https://doi.org/10.1111/j.1939-165X.2008.00104.x

Sakaguchi, S., 2000. Regulatory T cells: key controllers of immunologic self-tolerance. Cell 101, 455–458. https://doi.org/10.1016/S0092-8674(00)80856-9

Sakaguchi, S., Yamaguchi, T., Nomura, T., Ono, M., 2008. Regulatory T Cells and Immune Tolerance. Cell 133, 775–787. https://doi.org/10.1016/j.cell.2008.05.009

Sakai, K., Maeda, S., Yamada, Y., Chambers, J.K., Uchida, K., Nakayama, H., 82

Yonezawa, T., Matsuki, N., 2018. Association of tumour-infiltrating regulatory T cells with adverse outcomes in dogs with malignant tumours. Veterinary and Comparative Oncology 1, 1–7. https://doi.org/10.1111/vco.12383

Salama, P., Phillips, M., Grieu, F., Morris, M., Zeps, N., Joseph, D., Platell, C., Iacopetta, B., 2009. Tumor-infiltrating FOXP3+ T regulatory cells show strong prognostic significance in colorectal cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27, 186–92. https://doi.org/10.1200/JCO.2008.18.7229

Schlotter, Y.M., Veenhof, E.Z., Brinkhof, B., Rutten, V.P.M.G., Spee, B., Willemse, T., Penning, L.C., 2009. A GeNorm algorithm-based selection of reference genes for quantitative real-time PCR in skin biopsies of healthy dogs and dogs with atopic dermatitis. Veterinary Immunology and Immunopathology 129, 115–118. https://doi.org/10.1016/j.vetimm.2008.12.004

Schott, M., 2006. Immunesurveillance by dendritic cells: Potential implication for immunotherapy of endocrine cancers. Endocrine-Related Cancer 13, 779–795. https://doi.org/10.1677/erc.1.01133

Schubert, K., Polte, T., Bönisch, U., Schader, S., Holtappels, R., Hildebrandt, G., Lehmann, J., Simon, J.C., Anderegg, U., Saalbach, A., 2011. Thy-1 (CD90) regulates the extravasation of leukocytes during inflammation. European Journal of Immunology 41, 645–656. https://doi.org/10.1002/eji.201041117

Shang, B., Liu, Y., Jiang, S., Liu, Y., 2015. Prognostic value of tumor-infiltrating FoxP3+ regulatory T cells in cancers: a systematic review and meta-analysis. Scientific Reports 5, 15179. https://doi.org/10.1038/srep15179

Shi, S., Wang, L., Wang, G., Guo, Z., Wei, M., Meng, Y., 2013. B7-H1 Expression Is Associated with Poor Prognosis in Colorectal Carcinoma and Regulates the Proliferation and Invasion of HCT116 Colorectal Cancer Cells 8, 1–11. https://doi.org/10.1371/journal.pone.0076012

Shortman, K., Naik, S.H., 2007. Steady-state and inflammatory dendritic-cell development. Nature Reviews Immunology 7, 19–30. https://doi.org/10.1038/nri1996

Spangler, W.L., Culbertson, M.R., Kass, P.H., 1994. Primary Mesenchymal (Nonangiomatous/Nonlymphomatous) Neoplasms Occurring in the Canine Spleen: 83

Anatomic Classification, Immunohistochemistry, and Mitotic Activity Correlated with Patient Survival. Veterinary Pathology 31, 37–47. https://doi.org/10.1177/030098589403100105

Steinman, R.M., Hawiger, D., Nussenzweig, M.C., 2003. Tolerogenic dendritic cells. Annual review of immunology 21, 685–711. https://doi.org/10.1146/annurev.immunol.21.120601.141040

Strominger, J.L., 2010. An Alternative Path for Antigen Presentation: Group 1 CD1 Proteins. The Journal of Immunology 184, 3303–3305. https://doi.org/10.4049/jimmunol.1090008

Tagawa, M., Maekawa, N., Konnai, S., Takagi, S., 2016. Evaluation of costimulatory molecules in peripheral blood lymphocytes of canine patients with histiocytic sarcoma. PLoS ONE 11. https://doi.org/10.1371/journal.pone.0150030

Taylor, D.O.N., Dorn, C.R., Luis, O.H., 1969. Morphologic and Biologic Characteristics of the Canine Cutaneous Histiocytoma. Cancer Research 29, 83–92.

Tokuno, K., Hazama, S., Yoshino, S., Yoshida, S., Oka, M., 2009. Increased prevalence of regulatory T-cells in the peripheral blood of patients with gastrointestinal cancer. Anticancer Research 29, 1527–1532.

Tominaga, M., Horiuchi, Y., Ichikawa, M., Yamashita, M., Okano, K., Jikumaru, Y., Nariai, Y., Kadosawa, T., 2010. Flow Cytometric Analysis of Peripheral Blood and Tumor-Infiltrating Regulatory T Cells in Dogs with Oral Malignant Melanoma. Journal of Veterinary Diagnostic Investigation 22, 438–441. https://doi.org/10.1177/104063871002200317

Tsai, S., Sutherland-Smith, J., Burgess, K., Ruthazer, R., Sato, A., 2012. Imaging characteristics of intrathoracic histiocytic sarcoma in dogs. Veterinary Radiology and Ultrasound 53, 21–27. https://doi.org/10.1111/j.1740-8261.2011.01863.x

Voskoboinik, I., Whisstock, J.C., Trapani, J.A., 2015. Perforin and granzymes: Function, dysfunction and human pathology. Nature Reviews Immunology 15, 388–400. https://doi.org/10.1038/nri3839

Wang, W., Hodkinson, P., McLaren, F., MacKinnon, A., Wallace, W., Howie, S., Sethi, T., 2012. Small cell lung cancer tumour cells induce regulatory T lymphocytes, and patient survival correlates negatively with FOXP3+ cells in tumour infiltrate. International Journal of Cancer 131. https://doi.org/10.1002/ijc.27613

Whiteside, T.L., 2015. The role of regulatory T cells in cancer immunology. ImmunoTargets and therapy 4, 159–71. https://doi.org/10.2147/ITT.S55415

Wolf, A.M., Wolf, D., Steurer, M., Wolf, A.M., Wolf, D., Steurer, M., Gastl, G., Gunsilius, E., Grubeck-loebenstein, B., 2003. Increase of Regulatory T Cells in the Peripheral Blood of Cancer Patients. Clinical Cancer Research. 9:2, 606–612.

Zhang, X., Izikson, L., Liu, L., Weiner, H.L., 2001. Activation of CD25+CD4+ Regulatory T Cells by Oral Antigen Administration. The Journal of Immunology 167, 4245–4253. https://doi.org/10.4049/jimmunol.167.8.4245

Appendices

Appendix 1. Consent form

The Queen’s veterinary School Hospital University of Cambridge Madingley Road Cambridge CB3 0ES

Evaluation of T cell profiles in peripheral blood, lymph node and tumour in Flatcoated Retrievers affected by histiocytic sarcoma

Histiocytic sarcoma is a highly aggressive tumour that affects Flatcoated Retrievers among other breeds. The most common localization is within the soft tissue of the limbs, often surrounding the joints, causing swelling of the limb and lameness. A less common localization is visceral, and the spleen is often affected. Both forms sadly carry a very poor prognosis. We are looking to improve future treatment options for Flatcoated Retrievers affected by histiocytic sarcoma by investigating the relationship between the immune system of the patients and the tumour. In this project we will be concentrating on identifying the role of regulatory T cells in the development and progression of histiocytic sarcoma. These specific cells play a fundamental role in controlling levels of immune response. By doing so they avoid harmful and excessive auto-immune reactions. Over the past decades studies in human oncology have shown that these cells decrease anti-tumoural activity of the immune system, allowing the tumour to grow and spread. It has been demonstrated in previous studies that histiocytic sarcoma in Flatcoated Retrievers have a prominent population of regulatory T cells. Through analysis blood and tumour samples we are looking to investigate the role played by these cells in histiocytic sarcoma. A better understanding of this role will allow us to comprehend the relationship between immune system and cancer and help identify possible immunotherapy targets for future treatment.

We therefore seek your permission to perform flow cytometry on tumour & blood samples collected as part of the routine clinical management of your dog.

This study has been approved by the Department’s Ethics and Welfare committee.

Patient name: Owner consent: I have read the above the information and agree that flow cytometry is performed on the collected tumour and or blood sample. Name (capital letters):______Signature: ______Date (day/month/year): ______

Appendices

Appendix 2. Summary of cases selected for immunohistochemistry analysis

Case Number Age at diagnosis Sex Localisation

1 FC00/114 8y6m M thigh 2 FC02/108A 9y F shoulder 3 FC05/140B 8y M right brachium 4 FC06/003C 7y F stifle 5 FC06/020 6y7m F left hind limb 6 FC06/131 9y10m M stifle

7 FC07/055 11y F right hip region 8 FC08/004 10y M mid brachium 9 FC08/019A 9y6m M left elbow 10 FC08/089A 7y M left shoulder 11 FC09/072 7y5m F foreleg

12 FC09/100B 10y F shoulder 13 FC11/034A 10y8m M carpus 14 FC11/052B 8y3m F neck, dorsum

15 FC11/063 8y2m F axilla 16 FC11/072 9y M right shoulder 17 FC12/027A 7y6m F left elbow 18 FC12/038B 8y M right elbow 19 FC12/065E 8y7m M right elbow 20 FC12/093 6y M foreleg 21 FC13/036 8y1m F groin 22 FC13/062 10y10m M prescapular area

23 FC13/089 10y4m M interdigital 24 FC14/017B 7y11m M left shoulder 25 FC15/003B 9y10m F flank 26 FC15/035 8y F left antebrachium

27 FC17/001A 10y F shoulder

Appendices

Appendix 3. Summary of cases selected for flow cytometry analysis

Case Age Sex Number Affected 1 8y M 2 10y6m F 3 8y M 4 6y6m M 5 5y9m M 6 x F 7 9y10m M 8 7y6m M Control group 1 9y F 2 10y7m F 3 12y F 4 8y F 5 11y F 6 7y6m F 7 7y6m M 8 12y M 9 9y M 10 9y F 11 9y M

Appendices

Appendix 4. Canine PD-1 mRNA sequence (AB898677.1 top) aligned against human PD-1 mRNA sequence (NM_005018.2 bottom) using Matcher from the EMBOSS suite of programmes. The exon-exon boundaries are indicated by blue and black text. The start and stop codon are indicated by green and red text respectively. The positions of the forward and reverse primers are underlined. They span the predicted exon 2-3 boundary.

10 20 30 40 50 CACCTCTGCGGGAGCCGCCGGGGGAGGCGAGCAGGCGGGCTGGC--GCTC :::::: :: :::: : :: : ::::: ::: : :: :: :::: CACCTCCGCCTGAGCAGT-GGAGAAGGCG-GCACTCTGGTGGGGCTGCTC 20 30 40 50 60

60 70 80 90 100 CGGGCATGGGGAGCCGGCGGGGGCCCTGGCCGCTCGTCTGGGCCGTGCTG : :::::: :: :: : :: ::::::::: :::::::::: ::::: CAGGCATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTA 70 80 90 100 110

110 120 130 140 150 CAGCTGGGCTGGTGGCCAGGATGGCTCCTAGACTCCCCTGACAGGCCCTG :: ::::::::: ::::::::::: :: :::::::::: ::::::::::: CAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTG 120 130 140 150 160

160 170 180 190 200 GAGCCCGCTCACCTTCTCCCCGGCGCAGCTCACGGTGCAGGAGGGAGAGA :: ::: : :::::::::::: :: : :::: :::: :: :: :: : GAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACA 170 180 190 200 210

210 220 230 240 250 ACGCCACGTTCACCTGCAGCCTGGCCGACATCCCCGACAGCTTCGTGCTC ::::::: :::::::::::: : :: ::: : :: ::::::::::: ACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTA 220 230 240 250 260

260 270 280 290 300 AACTGGTACCGCCTGAGCCCCCGCAACCAGACGGACAAGCTGGCCGCCTT :::::::::::: :::::::: :::::::::::::::::::::::::::: AACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTT 270 280 290 300 310

310 320 330 340 350 CCAGGAGGACCGCATCGAGCCGGGCCGGGACAGGCGCTTCCGCGTCACGC :: :::::::::: : :::: :::: :::: : :::::::: ::::: : CCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACAC 320 330 340 350 360

Appendices

360 370 380 390 400 GGCTGCCCAACGGGCGGGACTTCCACATGAGCATCGTCGCTGCGCGCCTC :::::::::::::: ::::::::::::::: : ::: :: :: : : AACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGC 370 380 390 400 410

410 420 430 440 450 AACGACAGCGGCATCTACCTGTGCGGGGCCATCTACCTGCCCCCCAACAC :: :::::::::: :::::: :: :::::::::: :::: ::::::: : AATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGC 420 430 440 450 460

460 470 480 490 500 ACAGATCAACGAGAGTCCCCGCGCAGAGCTCTCCGTGACGGAGAGAACCC :::::::: ::::: : :: ::::::::: ::::: ::::::: GCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGG 470 480 490 500 510

510 520 530 540 550 TGGAGCCCCCCACACAGAGCCCCAGCCCCCCACCCAGACTCAGCGGCCAG :: :::::: :::::::::: ::::::: : ::::::: CAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAG 520 530 540 550 560

560 570 580 590 600 TTGCAGGGGCTGGTCATCGGCGTCACGAGCGTGCTGGTGGGTGTCCTGCT :: :: ::::: : :: ::: : ::: ::: :::: : :: TTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGG----CAGC- 570 580 590 600

610 620 630 640 650 ACTGCTGCTGCTGACCTGGGTCCTGGCCGCTGTCTTCCCCAGGGCCACCC ::: ::::::: :::::::::::::: ::: : :: ::::: : : -CTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCAC 610 620 630 640 650

660 670 680 690 700 GAGGTGCCTGTGTGTGCGGGAGCGAGGACGAGCCTCTGAAGGAGGGCCCC :::: : : : :: :: : : :::: :::::::::: :::: GAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCC 660 670 680 690 700

710 720 730 740 750 GATGCAGCGCCCGTCTTCACCCTGGACTACGGGGAGCTGGACTTCCAGTG :: : ::: :: ::: : ::::::: ::::::::::: :::::::: TCAGCCGTGCCTGTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTG 710 720 730 740 750

760 770 780 790 800 GCGAGAGAAGACGCCGGAGCCCCCGGCGCCCTGTGCCCCGGAGCAGACCG :::::::::::: ::::::::::: : :::::::: ::: :::::::: : GCGAGAGAAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGG 760 770 780 790 800

810 820 830 840 AGTATGCCACCATCGTCTTCCCGGGCAGGCCGG------CGTCCCCGGGC ::::::::::::: ::::: :: :: : :: : ::::: : : AGTATGCCACCATTGTCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCC 810 820 830 840 850

Appendices

850 860 870 880 890 CGCAGGGCCTCGGCCAGCAGCCTGCAGGGAGCCCAGCCTCCGAGCCCCGA ::::::: ::: :: : ::: : : : :::::::: : ::: :: :: CGCAGGGGCTCAGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGA 860 870 880 890 900

900 910 920 930 940 GGACGGACCCGGCCTGTGGCCCCTCTGACCGGCCGCCTCCGCTGGCCCAT ::: :::: : :: ::::::::::::::::: ::: :: :: : GGATGGACACTGCTCTTGGCCCCTCTGACCGGCTTCCTTGGCCA-CCAGT 910 920 930 940 950

950 960 970 980 990 GTCCTGCAGACTGTCCACCAGGAGCCCAGC---GGGCACCTCCCCTGAAG :: :::::::: ::::::: :::::: : : ::: ::: : : :: GTTCTGCAGACCCTCCACCATGAGCCCGGGTCAGCGCATTTCCTCAGGAG 960 970 980 990 1000

1000 1010 1020 1030 CAGCGAGTAGTGGGG-GGCCGGGG--GGCTGCTCCGGGGCTGAGC--CC- ::: : :: : : :::: : ::: : : :::::::::: :: AAGCAGGCAGGGTGCAGGCCATTGCAGGCCGTCCAGGGGCTGAGCTGCCT 1010 1020 1030 1040 1050

1040 1050 1060 1070 ----GTGACCCCCGCT---GCCTGTGGCAGCCCT---CATGGCTCTCCTC : :::: ::: ::::: : :: : :: :: : : : GGGGGCGACCGGGGCTCCAGCCTGCACCTGCACCAGGCACAGCCCCAC-C 1060 1070 1080 1090 1100

1080 1090 1100 1110 1120 TCTCGACTCACGAC-CTGGGGGCAGCGGGTGCCCCTGTCCC---TGCCCC : :::::: : : : : :: : : :::: : : :: : : ACAGGACTCATGTCTCAATGCCCACAGTGAGCCCAGGCAGCAGGTGTCAC 1110 1120 1130 1140 1150

1130 1140 1150 1160 1170 TGTCCCTCTCCGGGGCTGTGCCTGGGGAAAGGAGGCTCGCCCAGGACC-G ::::: :: : ::: : ::: : : : : :: :::: :: : CGTCCC-CTACAGGGA-GGGCCAGATGCA--GTCACTGCTTCAGGTCCTG 1160 1170 1180 1190 1200

1180 1190 1200 1210 CAGGCCCGGCCC--CCTGAG-CCAGATGG--GCCCCCCCGCCCCGATTGC : :: : : : :::: : :::: : : : : :: : ::: CCAGCACAGAGCTGCCTGCGTCCAGCTCCCTGAATCTCTGCTGCTGCTGC 1210 1220 1230 1240 1250

1220 1230 1240 1250 1260 TGCCCCGTGCTCCCACA-CGTGCCTCCCTGGG--GAAGGGGTCCAGTGCC ::: : :::: : : : ::: ::: ::: ::::: : : ::: : TGCTGC-TGCTGCTGCTGCCTGCGGCCCGGGGCTGAAGGCG--CCGTGGC 1260 1270 1280 1290

Appendices

1270 1280 1290 1300 GCGGCC-----CCCCGG------CTGCCTGCATGTGGGGACACAGGGACA : ::: :::::: ::::::: : ::::: : : CCTGCCTGACGCCCCGGAGCCTCCTGCCTGAACTTGGGGGCTGGTTGGAG 1300 1310 1320 1330 1340

1310 1320 1330 1340 1350 CTGGCCAGAGACCCCGCCGGGGACGGCCTGCTGAGGTCCCCTCTAGCCCA ::::: :: : ::: :: :::: : :: : :: : ATGGCCTTGGAGCA-GCCAAGGTGCCCCTG--GCAGTGGCATC-----CC 1350 1360 1370 1380 1390

1360 1370 1380 1390 1400 GAAACCCAGCCCAGCCCGGGGGTCCCACCCTGGGGCCGGGGCCCCGGAGC ::::: :::: : :: :: :::: :::: :: :::: GAAAC---GCCCTGGACGCAGGGCCCAAGACTGGGCACAGGAGTGGGAGG 1400 1410 1420 1430

1410 1420 1430 1440 TCCTCTTG--TGGGGAGGCCTGGGGA---ATC-GCTCCTGGCAGGGC-AG : : : :::::: :: ::: ::: ::::: ::::: : :: TACATGGGGCTGGGGACTCCCCAGGAGTTATCTGCTCCCTGCAGGCCTAG 1440 1450 1460 1470 1480

AG :: AG

Appendices

Appendix 5.

Canine PD-L1 mRNA sequence (AB898678.1 top) aligned against human PD-L1 mRNA sequence (NM_014143.3 bottom) using Matcher from the EMBOSS suite of programmes. The exon-exon boundaries are indicated by blue and black text. The start and stop codon are indicated by green and red text respectively. The positions of the forward and reverse primers are underlined. They span the predicted exon 5-7 boundary.

CLUSTAL O(1.2.4) multiple sequence alignment

AB898678.1 ------XM_005615932.3 ------XM_005615933.3 GAGCACCTCGGTGGAGGCGGCGGGGCTGTCCCGGAGCCCAGCACTGCCCGGCGCTCATCC XM_022421251.1 ------XM_022421407.1 ------XM_022421286.1 ------XM_022421319.1 ------XM_005615931.3 ------XM_005615934.3 ------NM_001314029.1 ------NR_052005.1 ------NM_014143.3 ------NM_001267706.1 ------

AB898678.1 ------XM_005615932.3 ------XM_005615933.3 CGCATCCCGGTCCTCTCCGCCCTCCGCCTCCGGGGCTTCGTCTCCGTCGTCCGTTTCCCC XM_022421251.1 ------XM_022421407.1 ------XM_022421286.1 ------XM_022421319.1 ------XM_005615931.3 ------XM_005615934.3 ------NM_001314029.1 ------NR_052005.1 ------NM_014143.3 ------NM_001267706.1 ------

AB898678.1 ------XM_005615932.3 ------XM_005615933.3 GGCGTTGTCTTCCTGCCCAGGGACACCAGGAGCGCTGTCCACGTAGCTGGATTCGGAGTT XM_022421251.1 ----GCTGGGATGCTGACAGGGACACCAGGAGCGCTGTCCACGTAGCTGGATTCGGAGTT XM_022421407.1 ------XM_022421286.1 --AAGTGCTCGCGGAGCCTGGGACACCAGGAGCGCTGTCCACGTAGCTGGATTCGGAGTT XM_022421319.1 ---AGTGCTCGCGGAGCCTGGGACACCAGGAGCGCTGTCCACGTAGCTGGATTCGGAGTT XM_005615931.3 ------XM_005615934.3 ------NM_001314029.1 ------NR_052005.1 ------NM_014143.3 ------NM_001267706.1 ------

Appendices

AB898678.1 ------XM_005615932.3 ------XM_005615933.3 TTGAAAAGGCCGGTGTGATTTGATGAGCACGGAGTCCTCCCCTTCCCTGAAAGGCAGCTC XM_022421251.1 TTGAAAAGGCCGGTGTGATTTGATGAGCACGGAGTCCTCCCCTTCCCTGAAAGGCAGCTC XM_022421407.1 ------XM_022421286.1 TTGAAAAGGCCGGTGTGATTTGATGAGCACGGAGTCCTCCCCTTCCCTGAAAGGCAGCTC XM_022421319.1 TTGAAAAGGCCGGTGTGATTTGATGAGCACGGAGTCCTCCCCTTCCCTGAAAGGCAGCTC XM_005615931.3 ------XM_005615934.3 ------NM_001314029.1 ------NR_052005.1 ------NM_014143.3 ------NM_001267706.1 ------

AB898678.1 ------XM_005615932.3 ------XM_005615933.3 GTGTCCCCGCTGCGGTGAGCCGTGAGTTCTAGCCAGAACTCGGTCAGGGGTGCCCTGTGA XM_022421251.1 GTGTCCCCGCTGCGGTGAGCCGTGAGTTCTAGCCAGAACTCGGTCAGGGGTGCCCTGTGA XM_022421407.1 ------XM_022421286.1 GTGTCCCCGCTGCGGTGAGCCGTGAGTTCTAGCCAGAACTCGGTCAGGGGTGCCCTGTGA XM_022421319.1 GTGTCC------XM_005615931.3 ------XM_005615934.3 ------NM_001314029.1 ------NR_052005.1 ------NM_014143.3 ------NM_001267706.1 ------

AB898678.1 ------XM_005615932.3 ------XM_005615933.3 AAACCGAAAACATTCGTTATACCGAGAGGGAGGATCTGAAACAAAGTTCTGCTGAGCTCT XM_022421251.1 AAACCGAAAACATTCGTTATACCGAGAGGGAGGATCTGAAACAAAGTTCTGCTGAGCTCT XM_022421407.1 ------AAGTGCTCGCGGAGCCT XM_022421286.1 AAACCGAAAACATTCGTTATACCGAGAGGGAGGATCTGAAACAAAGTTCTGCTGAGCTCT XM_022421319.1 ------XM_005615931.3 ------XM_005615934.3 ------NM_001314029.1 ------NR_052005.1 ------NM_014143.3 ------NM_001267706.1 ------

AB898678.1 ------XM_005615932.3 ------ATCACATGCTACATCACAATGCCTACATTAT XM_005615933.3 AGGGAGGG------XM_022421251.1 AGGGAGGGAGGAATAGACAGGCCAGCAGAAGCAGTGTGTATGGAAGATGGAGTAACTGAG XM_022421407.1 GGGACACCAGG------AGCGCTGTCCACGTAGCTGGATTCGGAGTTTTGA-AAAGGCC XM_022421286.1 AGGGAGGGAGGAATAGACAGGCCAGCAGAAGCAGTGTGTATGGAAGATGGAGTAACTGAG XM_022421319.1 --CCGCTGCGGAATAGACAGGCCAGCAGAAGCAGTGTGTATGGAAGATGGAGTAACTGAG XM_005615931.3 ------GAGGCGGGAGG XM_005615934.3 ------NM_001314029.1 ------NR_052005.1 ------NM_014143.3 ------NM_001267706.1 ------

Appendices

AB898678.1 ------XM_005615932.3 TG-----ACCTCACTTCATCTTAGCACATAGGCATTTTCTTATTTCACATCACTACAAGA XM_005615933.3 ------XM_022421251.1 TCATCGAATGGTGGTGGAAGACCTCAAATGTCATTAGTTCAGTATGGCGTGAGAACGACT XM_022421407.1 GGTGTGATTTGATGAGCACGGAGTCCTCCCCTTCCCTGAAAGGCAGCTCGTGTCCCCGCT XM_022421286.1 TCATCGAATGGTGGTGGAAGACCTCAAATGTCATTAGTTCAGTATGGCGTGAGAACGACT XM_022421319.1 TCATCGAATGGTGGTGGAAGACCTCAAATGTCATTAGTTCAGTATGGCGTGAGAACGACT XM_005615931.3 CGCGAGGCGCGAGGCGCGAGGCGCCCGGTGCCGGGTGCTAGATGGAAGTGCTCGCGGAGC XM_005615934.3 GGGGGGGGGGGAGGGGCGCAGCCTCGACCTCTGGACCGAGGGCCGTGGCTGGGATGCTGA NM_001314029.1 ------NR_052005.1 ------NM_014143.3 ------NM_001267706.1 ------

AB898678.1 ------XM_005615932.3 AGGAGCATTGGGATACTAAGCTTAGAAGGAACCTCTGCTCTCTTGAGATCCTGATGCGGA XM_005615933.3 -AGAGCATTGGGATACTAAGCTTAGAAGGAACCTCTGCTCTCTTGAGATCCTGATGCGGA XM_022421251.1 CAGAGCATTGGGATACTAAGCTTAGAAGGAACCTCTGCTCTCTTGAGATCCTGATGCGGA XM_022421407.1 GCGAGCATTGGGATACTAAGCTTAGAAGGAACCTCTGCTCTCTTGAGATCCTGATGCGGA XM_022421286.1 CAGAGCATTGGGATACTAAGCTTAGAAGGAACCTCTGCTCTCTTGAGATCCTGATGCGGA XM_022421319.1 CAGAGCATTGGGATACTAAGCTTAGAAGGAACCTCTGCTCTCTTGAGATCCTGATGCGGA XM_005615931.3 CTGAGCATTGGGATACTAAGCTTAGAAGGAACCTCTGCTCTCTTGAGATCCTGATGCGGA XM_005615934.3 CAGAGCATTGGGATACTAAGCTTAGAAGGAACCTCTGCTCTCTTGAGATCCTGATGCGGA NM_001314029.1 ------GGCGCAACGCTGAGCAGCTGGCGCG-- NR_052005.1 ------GGCGCAACGCTGAGCAGCTGGCGCG-- NM_014143.3 ------GGCGCAACGCTGAGCAGCTGGCGCG-- NM_001267706.1 ------

AB898678.1 ------AGCGGCCCCGACACCGCGTGCAGCACCTCCCGCCCGGCCGCCGCCAGCTC XM_005615932.3 TTAGGAAAACGCATGGACCTCTGGGGGTGTCATCTCTTAAGTCTTAGCACCAGATGGATG XM_005615933.3 TTAGGAAAACGCATGGACCTCTGGGGGTGTCATCTCTTAAGTCTTAGCACCAGATGGATG XM_022421251.1 TTAGGAAAACGCATGGACCTCTGGGGGTGTCATCTCTTAAGTCTTAGCACCAGATGGATG XM_022421407.1 TTAGGAAAACGCATGGACCTCTGGGGGTGTCATCTCTTAAGTCTTAGCACCAGATGGATG XM_022421286.1 TTAGGAAAACGCATGGACCTCTGGGGGTGTCATCTCTTAAGTCTTAGCACCAGATGGATG XM_022421319.1 TTAGGAAAACGCATGGACCTCTGGGGGTGTCATCTCTTAAGTCTTAGCACCAGATGGATG XM_005615931.3 TTAGGAAAACGCATGGACCTCTGGGGGTGTCATCTCTTAAGTCTTAGCACCAGATGGATG XM_005615934.3 TTAGGAAAACGCATGGACCTCTGGGGGTGTCATCTCTTAAGTCTTAGCACCAGATGGATG NM_001314029.1 TCCCGCGCGGCCCCAGTTCTGCGCAGCT--TCCCGAGGCTCCGCACCAGCCGCGCTTCTG NR_052005.1 TCCCGCGCGGCCCCAGTTCTGCGCAGCT--TCCCGAGGCTCCGCACCAGCCGCGCTTCTG NM_014143.3 TCCCGCGCGGCCCCAGTTCTGCGCAGCT--TCCCGAGGCTCCGCACCAGCCGCGCTTCTG NM_001267706.1 ------

AB898678.1 CCCGCCAGCAGGTCACTTCAGAACGATGAGAATGTTTAGTGTCTTTACATTCATGGCCTA XM_005615932.3 ATCTTACGGTGGTCACTTCAGAACGATGAGAATGTTTAGTGTCTTTACATTCATGGCCTA XM_005615933.3 ATCTTACGGTGGTCACTTCAGAACGATGAGAATGTTTAGTGTCTTTACATTCATGGCCTA XM_022421251.1 ATCTTACGGTGGTCACTTCAGAACGATGAGAATGTTTAGTGTCTTTACATTCATGGCCTA XM_022421407.1 ATCTTACGGTGGTCACTTCAGAACGATGAGAATGTTTAGTGTCTTTACATTCATGGCCTA XM_022421286.1 ATCTTACGGTGGTCACTTCAGAACGATGAGAATGTTTAGTGTCTTTACATTCATGGCCTA XM_022421319.1 ATCTTACGGTGGTCACTTCAGAACGATGAGAATGTTTAGTGTCTTTACATTCATGGCCTA XM_005615931.3 ATCTTACGGTGGTCACTTCAGAACGATGAGAATGTTTAGTGTCTTTACATTCATGGCCTA XM_005615934.3 ATCTTACGGTGGTCACTTCAGAACGATGAGAATGTTTAGTGTCTTTACATTCATGGCCTA NM_001314029.1 TCCGCCTGCAGGGCATTCCAGAAAGATGAGGATATTTGCTGTCTTTATATTCATGACCTA NR_052005.1 TCCGCCTGCAGGGCATTCCAGAAAGATGAGGATATTTGCTGTCTTTATATTCATGACCTA NM_014143.3 TCCGCCTGCAGGGCATTCCAGAAAGATGAGGATATTTGCTGTCTTTATATTCATGACCTA NM_001267706.1 ------

Appendices

AB898678.1 CTGCCATTTGCTAAAAGCATTTACGATCACAGTTTCTAAGGACCTGTATGTGGTAGAGTA XM_005615932.3 CTGCCATTTGCTAAAAGCATTTACGATCACAGTTTCTAAGGACCTGTATGTGGTAGAGTA XM_005615933.3 CTGCCATTTGCTAAAAGCATTTACGATCACAGTTTCTAAGGACCTGTATGTGGTAGAGTA XM_022421251.1 CTGCCATTTGCTAAAAGCATTTACGATCACAGTTTCTAAGGACCTGTATGTGGTAGAGTA XM_022421407.1 CTGCCATTTGCTAAAAGCATTTACGATCACAGTTTCTAAGGACCTGTATGTGGTAGAGTA XM_022421286.1 CTGCCATTTGCTAAAAGCATTTACGATCACAGTTTCTAAGGACCTGTATGTGGTAGAGTA XM_022421319.1 CTGCCATTTGCTAAAAGCATTTACGATCACAGTTTCTAAGGACCTGTATGTGGTAGAGTA XM_005615931.3 CTGCCATTTGCTAAAAGCATTTACGATCACAGTTTCTAAGGACCTGTATGTGGTAGAGTA XM_005615934.3 CTGCCATTTGCTAAAAGCATTTACGATCACAGTTTCTAAGGACCTGTATGTGGTAGAGTA NM_001314029.1 CTGGCATTTGCTGAACGCATTTACTGTCACGGTTCCCAAGGACCTATATGTGGTAGAGTA NR_052005.1 CTGGCATTTGCTGAACGCATTTACTGTCACGGTTCCCAAGGACCTATATGTGGTAGAGTA NM_014143.3 CTGGCATTTGCTGAACGCATTTACTGTCACGGTTCCCAAGGACCTATATGTGGTAGAGTA NM_001267706.1 ------

AB898678.1 TGGTGGCAATGTGACAATGGAATGCAAATTCCCGGTGGAAAAACAGTTAAACTTGTTTGC XM_005615932.3 TGGTGGCAATGTGACAATGGAATGCAAATTCCCGGTGGAAAAACAGTTAAACTTGTTTGC XM_005615933.3 TGGTGGCAATGTGACAATGGAATGCAAATTCCCGGTGGAAAAACAGTTAAACTTGTTTGC XM_022421251.1 TGGTGGCAATGTGACAATGGAATGCAAATTCCCGGTGGAAAAACAGTTAAACTTGTTTGC XM_022421407.1 TGGTGGCAATGTGACAATGGAATGCAAATTCCCGGTGGAAAAACAGTTAAACTTGTTTGC XM_022421286.1 TGGTGGCAATGTGACAATGGAATGCAAATTCCCGGTGGAAAAACAGTTAAACTTGTTTGC XM_022421319.1 TGGTGGCAATGTGACAATGGAATGCAAATTCCCGGTGGAAAAACAGTTAAACTTGTTTGC XM_005615931.3 TGGTGGCAATGTGACAATGGAATGCAAATTCCCGGTGGAAAAACAGTTAAACTTGTTTGC XM_005615934.3 TGGTGGCAATGTGACAATGGAATGCAAATTCCCGGTGGAAAAACAGTTAAACTTGTTTGC NM_001314029.1 TGGTAGCAATATGACAATTGAATGCAAATTCCCAGTAGAAAAACAATTAGACCTGGCTGC NR_052005.1 TGGTAGCAATATGACAATTGAATGCAAATTCCCAGTAGAAAAACAATTAGACCTGGCTGC NM_014143.3 TGGTAGCAATATGACAATTGAATGCAAATTCCCAGTAGAAAAACAATTAGACCTGGCTGC NM_001267706.1 ------

AB898678.1 ACTAATCGTCTACTGGGAAATGGAGGATAAAAAAATTATACAATTTGTGAATGGAAAGGA XM_005615932.3 ACTAATCGTCTACTGGGAAATGGAGGATAAAAAAATTATACAATTTGTGAATGGAAAGGA XM_005615933.3 ACTAATCGTCTACTGGGAAATGGAGGATAAAAAAATTATACAATTTGTGAATGGAAAGGA XM_022421251.1 ACTAATCGTCTACTGGGAAATGGAGGATAAAAAAATTATACAATTTGTGAATGGAAAGGA XM_022421407.1 ACTAATCGTCTACTGGGAAATGGAGGATAAAAAAATTATACAATTTGTGAATGGAAAGGA XM_022421286.1 ACTAATCGTCTACTGGGAAATGGAGGATAAAAAAATTATACAATTTGTGAATGGAAAGGA XM_022421319.1 ACTAATCGTCTACTGGGAAATGGAGGATAAAAAAATTATACAATTTGTGAATGGAAAGGA XM_005615931.3 ACTAATCGTCTACTGGGAAATGGAGGATAAAAAAATTATACAATTTGTGAATGGAAAGGA XM_005615934.3 ACTAATCGTCTACTGGGAAATGGAGGATAAAAAAATTATACAATTTGTGAATGGAAAGGA NM_001314029.1 ACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTATTCAATTTGTGCATGGAGAGGA NR_052005.1 ACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTATTCAATTTGTGCATGGAGAGGA NM_014143.3 ACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTATTCAATTTGTGCATGGAGAGGA NM_001267706.1 ------

AB898678.1 AGACCTGAAAGTTCAGCACAGCAGCTACAGCCAGAGGGCTCAGCTATTGAAGGACCAGCT XM_005615932.3 AGACCTGAAAGTTCAGCACAGCAGCTACAGCCAGAGGGCTCAGCTATTGAAGGACCAGCT XM_005615933.3 AGACCTGAAAGTTCAGCACAGCAGCTACAGCCAGAGGGCTCAGCTATTGAAGGACCAGCT XM_022421251.1 AGACCTGAAAGTTCAGCACAGCAGCTACAGCCAGAGGGCTCAGCTATTGAAGGACCAGCT XM_022421407.1 AGACCTGAAAGTTCAGCACAGCAGCTACAGCCAGAGGGCTCAGCTATTGAAGGACCAGCT XM_022421286.1 AGACCTGAAAGTTCAGCACAGCAGCTACAGCCAGAGGGCTCAGCTATTGAAGGACCAGCT XM_022421319.1 AGACCTGAAAGTTCAGCACAGCAGCTACAGCCAGAGGGCTCAGCTATTGAAGGACCAGCT XM_005615931.3 AGACCTGAAAGTTCAGCACAGCAGCTACAGCCAGAGGGCTCAGCTATTGAAGGACCAGCT XM_005615934.3 AGACCTGAAAGTTCAGCACAGCAGCTACAGCCAGAGGGCTCAGCTATTGAAGGACCAGCT NM_001314029.1 AGACCTGAAGGTTCAGCATAGTAGCTACAGACAGAGGGCCCGGCTGTTGAAGGACCAGCT NR_052005.1 AGACCTGAAGGTTCAGCATAGTAGCTACAGACAGAGGGCCCGGCTGTTGAAGGACCAGCT NM_014143.3 AGACCTGAAGGTTCAGCATAGTAGCTACAGACAGAGGGCCCGGCTGTTGAAGGACCAGCT NM_001267706.1 --GGCGCAACGCTG------AGCAGCTGGCGCGTCCCGCGCGGCCCCAGTT * ** * * *** *** * * * * **** *

AB898678.1 CTTCTTGGGGAAGGCTGCGCTTCAGATCACAGATGTGAGATTGCAGGATGCAGGGGTTTA

Appendices

XM_005615932.3 CTTCTTGGGGAAGGCTGCGCTTCAGATCACAGATGTGAGATTGCAGGATGCAGGGGTTTA XM_005615933.3 CTTCTTGGGGAAGGCTGCGCTTCAGATCACAGATGTGAGATTGCAGGATGCAGGGGTTTA XM_022421251.1 CTTCTTGGGGAAGGCTGCGCTTCAGATCACAGATGTGAGATTGCAGGATGCAGGGGTTTA XM_022421407.1 CTTCTTGGGGAAGGCTGCGCTTCAGATCACAGATGTGAGATTGCAGGATGCAGGGGTTTA XM_022421286.1 CTTCTTGGGGAAGGCTGCGCTTCAGATCACAGATGTGAGATTGCAGGATGCAGGGGTTTA XM_022421319.1 CTTCTTGGGGAAGGCTGCGCTTCAGATCACAGATGTGAGATTGCAGGATGCAGGGGTTTA XM_005615931.3 CTTCTTGGGGAAGGCTGCGCTTCAGATCACAGATGTGAGATTGCAGGATGCAGGGGTTTA XM_005615934.3 CTTCTTGGGGAAGGCTGCGCTTCAGATCACAGATGTGAGATTGCAGGATGCAGGGGTTTA NM_001314029.1 CTCCCTGGGAAATGCTGCACTTCAGATCACAGATGTGAAATTGCAGGATGCAGGGGTGTA NR_052005.1 CTCCCTGGGAAATGCTGCACTTCAGATCACAGATGTGAAATTGCAGGATGCAGGGGTGTA NM_014143.3 CTCCCTGGGAAATGCTGCACTTCAGATCACAGATGTGAAATTGCAGGATGCAGGGGTGTA NM_001267706.1 CTGCGC--AGCTTCCCGAGGCTCCGCACCAGCCGCGCTTCTGTCCGCCTGCAGGGCATTC ** * * * ** * * * * * ******* *

AB898678.1 CTGCTGCTTGATCGGCTATGGCGGTGCTGACTACAAGCGGATTACTTTGAAAGTTCATGC XM_005615932.3 CTGCTGCTTGATCGGCTATGGCGGTGCTGACTACAAGCGGATTACTTTGAAAGTTCATGC XM_005615933.3 CTGCTGCTTGATCGGCTATGGCGGTGCTGACTACAAGCGGATTACTTTGAAAGTTCATGC XM_022421251.1 CTGCTGCTTGATCGGCTATGGCGGTGCTGACTACAAGCGGATTACTTTGAAAGTTCATGC XM_022421407.1 CTGCTGCTTGATCGGCTATGGCGGTGCTGACTACAAGCGGATTACTTTGAAAGTTCATGC XM_022421286.1 CTGCTGCTTGATCGGCTATGGCGGTGCTGACTACAAGCGGATTACTTTGAAAGTTCATGC XM_022421319.1 CTGCTGCTTGATCGGCTATGGCGGTGCTGACTACAAGCGGATTACTTTGAAAGTTCATGC XM_005615931.3 CTGCTGCTTGATCGGCTATGGCGGTGCTGACTACAAGCGGATTACTTTGAAAGTTCATGC XM_005615934.3 CTGCTGCTTGATCGGCTATGGCGGTGCTGACTACAAGCGGATTACTTTGAAAGTTCATGC NM_001314029.1 CCGCTGCATGATCAGCTATGGTGGTGCCGACTACAAGCGAATTACTGTGAAAGTCAATGC NR_052005.1 CCGCTGCATGATCAGCTATGGTGGTGCCGACTACAAGCGAATTACTGTGAAAGTCAATGC NM_014143.3 CCGCTGCATGATCAGCTATGGTGGTGCCGACTACAAGCGAATTACTGTGAAAGTCAATGC NM_001267706.1 CAGAAAGATGAGGATATTTGCTGTCTTTATATTCATGACCTACTGGCATTTGCTGAACGC * * *** * ** * * ** * * * **

AB898678.1 CCCGTACCGCAACATCAGCCAAAGAATTT---CTGTGGATCCTGTCACCTCTGAACATGA XM_005615932.3 CCCGTACCGCAACATCAGCCAAAGAATTT---CTGTGGATCCTGTCACCTCTGAACATGA XM_005615933.3 CCCGTACCGCAACATCAGCCAAAGAATTT---CTGTGGATCCTGTCACCTCTGAACATGA XM_022421251.1 CCCGTACCGCAACATCAGCCAAAGAATTT---CTGTGGATCCTGTCACCTCTGAACATGA XM_022421407.1 CCCGTACCGCAACATCAGCCAAAGAATTT---CTGTGGATCCTGTCACCTCTGAACATGA XM_022421286.1 CCCGTACCGCAACATCAGCCAAAGAATTT---CTGTGGATCCTGTCACCTCTGAACATGA XM_022421319.1 CCCGTACCGCAACATCAGCCAAAGAATTT---CTGTGGATCCTGTCACCTCTGAACATGA XM_005615931.3 CCCGTACCGCAACATCAGCCAAAGAATTT---CTGTGGATCCTGTCACCTCTGAACATGA XM_005615934.3 CCCGTACCGCAACATCAGCCAAAGAATTT---CTGTGGATCCTGTCACCTCTGAACATGA NM_001314029.1 CCCATACAACAAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGA NR_052005.1 CCCATACAACAAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGA NM_014143.3 CCCATACAACAAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGA NM_001267706.1 CCCATACAACAAAATCAACCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGA *** *** *** **** *********** ********* *****************

AB898678.1 ACTAATGTGTCAGGCTGAGGGTTACCCTGAGGCTGAAGTCATCTGGACAAGCAGTGACCA XM_005615932.3 ACTAATGTGTCAGGCTGAGGGTTACCCTGAGGCTGAAGTCATCTGGACAAGCAGTGACCA XM_005615933.3 ACTAATGTGTCAGGCTGAGGGTTACCCTGAGGCTGAAGTCATCTGGACAAGCAGTGACCA XM_022421251.1 ACTAATGTGTCAGGCTGAGGGTTACCCTGAGGCTGAAGTCATCTGGACAAGCAGTGACCA XM_022421407.1 ACTAATGTGTCAGGCTGAGGGTTACCCTGAGGCTGAAGTCATCTGGACAAGCAGTGACCA XM_022421286.1 ACTAATGTGTCAGGCTGAGGGTTACCCTGAGGCTGAAGTCATCTGGACAAGCAGTGACCA XM_022421319.1 ACTAATGTGTCAGGCTGAGGGTTACCCTGAGGCTGAAGTCATCTGGACAAGCAGTGACCA XM_005615931.3 ACTAATGTGTCAGGCTGAGGGTTACCCTGAGGCTGAAGTCATCTGGACAAGCAGTGACCA XM_005615934.3 ACTAATGTGTCAGGCTGAGGGTTACCCTGAGGCTGAAGTCATCTGGACAAGCAGTGACCA NM_001314029.1 ACTGACATGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCA NR_052005.1 ACTGACATGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCA NM_014143.3 ACTGACATGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCA NM_001267706.1 ACTGACATGTCAGGCTGAGGGCTACCCCAAGGCCGAAGTCATCTGGACAAGCAGTGACCA *** * ************** ***** **** **************************

AB898678.1 CCGAGTCCTGAGTGGCAAAACCACCATCACTAATTCCAATAGGGAAGAGAAGCTTTTCAA

Appendices

XM_005615932.3 CCGAGTCCTGAGTGGCAAAACCACCATCACTAATTCCAATAGGGAAGAGAAGCTTTTCAA XM_005615933.3 CCGAGTCCTGAGTGGCAAAACCACCATCACTAATTCCAATAGGGAAGAGAAGCTTTTCAA XM_022421251.1 CCGAGTCCTGAGTGGCAAAACCACCATCACTAATTCCAATAGGGAAGAGAAGCTTTTCAA XM_022421407.1 CCGAGTCCTGAGTGGCAAAACCACCATCACTAATTCCAATAGGGAAGAGAAGCTTTTCAA XM_022421286.1 CCGAGTCCTGAGTGGCAAAACCACCATCACTAATTCCAATAGGGAAGAGAAGCTTTTCAA XM_022421319.1 CCGAGTCCTGAGTGGCAAAACCACCATCACTAATTCCAATAGGGAAGAGAAGCTTTTCAA XM_005615931.3 CCGAGTCCTGAGTGGCAAAACCACCATCACTAATTCCAATAGGGAAGAGAAGCTTTTCAA XM_005615934.3 CCGAGTCCTGAGTGGCAAAACCACCATCACTAATTCCAATAGGGAAGAGAAGCTTTTCAA NM_001314029.1 TCAAGTCCTGAGTGGTAAGACCACCACCACCAATTCCAAGAGAGAGGAGAAGCTTTTCAA NR_052005.1 TCAAGTCCTGAGTGGAG------NM_014143.3 TCAAGTCCTGAGTGGTAAGACCACCACCACCAATTCCAAGAGAGAGGAGAAGCTTTTCAA NM_001267706.1 TCAAGTCCTGAGTGGTAAGACCACCACCACCAATTCCAAGAGAGAGGAGAAGCTTTTCAA * ************

AB898678.1 TGTGACCAGCACGCTGAACATCAATGCAACAGCTAATGAGATTTTCTACTGCACTTTTCA XM_005615932.3 TGTGACCAGCACGCTGAACATCAATGCAACAGCTAATGAGATTTTCTACTGCACTTTTCA XM_005615933.3 TGTGACCAGCACGCTGAACATCAATGCAACAGCTAATGAGATTTTCTACTGCACTTTTCA XM_022421251.1 TGTGACCAGCACGCTGAACATCAATGCAACAGCTAATGAGATTTTCTACTGCACTTTTCA XM_022421407.1 TGTGACCAGCACGCTGAACATCAATGCAACAGCTAATGAGATTTTCTACTGCACTTTTCA XM_022421286.1 TGTGACCAGCACGCTGAACATCAATGCAACAGCTAATGAGATTTTCTACTGCACTTTTCA XM_022421319.1 TGTGACCAGCACGCTGAACATCAATGCAACAGCTAATGAGATTTTCTACTGCACTTTTCA XM_005615931.3 TGTGACCAGCACGCTGAACATCAATGCAACAGCTAATGAGATTTTCTACTGCACTTTTCA XM_005615934.3 TGTGACCAGCACGCTGAACATCAATGCAACAGCTAATGAGATTTTCTACTGCACTTTTCA NM_001314029.1 TGTGACCAGCACACTGAGAATCAACACAACAACTAATGAGATTTTCTACTGCACTTTTAG NR_052005.1 ------NM_014143.3 TGTGACCAGCACACTGAGAATCAACACAACAACTAATGAGATTTTCTACTGCACTTTTAG NM_001267706.1 TGTGACCAGCACACTGAGAATCAACACAACAACTAATGAGATTTTCTACTGCACTTTTAG

AB898678.1 AAGATCAGGTCCTGAGGAAAACAATACTGCCGAGTTGGTCATCCCAGAACGACTGCCCGT XM_005615932.3 AAGATCAGGTCCTGAGGAAAACAATACTGCCGAGTTGGTCATCCCAGAACGACTGCCCGT XM_005615933.3 AAGATCAGGTCCTGAGGAAAACAATACTGCCGAGTTGGTCATCCCAGAACGACTGCCCGT XM_022421251.1 AAGATCAGGTCCTGAGGAAAACAATACTGCCGAGTTGGTCATCCCAGAACGACTGCCCGT XM_022421407.1 AAGATCAGGTCCTGAGGAAAACAATACTGCCGAGTTGGTCATCCCAGAACGACTGCCCGT XM_022421286.1 AAGATCAGGTCCTGAGGAAAACAATACTGCCGAGTTGGTCATCCCAGAACGACTGCCCGT XM_022421319.1 AAGATCAGGTCCTGAGGAAAACAATACTGCCGAGTTGGTCATCCCAGAACGACTGCCCGT XM_005615931.3 AAGATCAGGTCCTGAGGAAAACAATACTGCCGAGTTGGTCATCCCAGAACGACTGCCCGT XM_005615934.3 AAGATCAGGTCCTGAGGAAAACAATACTGCCGAGTTGGTCATCCCAGAACGACTGCCCGT NM_001314029.1 GAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAGGTAATATTCTGAA NR_052005.1 ---ATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAGAACTACCTCTGGC NM_014143.3 GAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAGAACTACCTCTGGC NM_001267706.1 GAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAGAACTACCTCTGGC ** ** ************* **** ** ** ************* *

AB898678.1 TCCAGCAAGTGA---GAGGACTCATTTCATGATTCTGGGACCTTTCCTGTTGCTTCTTGG XM_005615932.3 TCCAGCAAGTGA---GAGGACTCATTTCATGATTCTGGGACCTTTCCTGTTGCTTCTTGG XM_005615933.3 TCCAGCAAGTGA---GAGGACTCATTTCATGATTCTGGGACCTTTCCTGTTGCTTCTTGG XM_022421251.1 TCCAGCAAGTGA---GAGGACTCATTTCATGATTCTGGGACCTTTCCTGTTGCTTCTTGG XM_022421407.1 TCCAGCAAGTGA---GAGGACTCATTTCATGATTCTGGGACCTTTCCTGTTGCTTCTTGG XM_022421286.1 TCCAGCAAGTGA---GAGGACTCATTTCATGATTCTGGGACCTTTCCTGTTGCTTCTTGG XM_022421319.1 TCCAGCAAGTGA---GAGGACTCATTTCATGATTCTGGGACCTTTCCTGTTGCTTCTTGG XM_005615931.3 TCCAGCAAGTGA---GAGGACTCATTTCATGATTCTGGGACCTTTCCTGTTGCTTCTTGG XM_005615934.3 TCCAGCAAGTGA---GAGGACTCATTTCATGATTCTGGGACCTTTCCTGTTGCTTCTTGG NM_001314029.1 TGTGTCCATTAAAATATGTCTAACACTGTCCCCT--AGCACC--TAGCATGATGTCT--G NR_052005.1 ACATCCTCCAAATGAAAGGACTCACTT------NM_014143.3 ACATCCTCCAAATGAAAGGACTCACTTGGTAATTCTGGGAGCCATCTTATTATGCCTTGG NM_001267706.1 ACATCCTCCAAATGAAAGGACTCACTTGGTAATTCTGGGAGCCATCTTATTATGCCTTGG * * * *

AB898678.1 TGTAGTCCTGGCAGTCACTTTCTGTCTAAAAAAACATGGGAGAATGATGGATGTGGAAAA XM_005615932.3 TGTAGTCCTGGCAGTCACTTTCTGTCTAAAAAAACATGGGAGAATGATGGATGTGGAAAA

Appendices

XM_005615933.3 TGTAGTCCTGGCAGTCACTTTCTGTCTAAAAAAACATGGGAGAATGATGGATGTGGAAAA XM_022421251.1 TGTAGTCCTGGCAGTCACTTTCTGTCTAAAAAAACATGGGAGAATGATGGATGTGGAAAA XM_022421407.1 TGTAGTCCTGGCAGTCACTTTCTGTCTAAAAAAACATGGGAGAATGATGGATGTGGAAAA XM_022421286.1 TGTAGTCCTGGCAGTCACTTTCTGTCTAAAAAAACATGGGAGAATGATGGATGTGGAAAA XM_022421319.1 TGTAGTCCTGGCAGTCACTTTCTGTCTAAAAAAACATGGGAGAATGATGGATGTGGAAAA XM_005615931.3 TGTAGTCCTGGCAGTCACTTTCTGTCTAAAAAAACATGGGAGAATGATGGATGTGGAAAA XM_005615934.3 TGTAGTCCTGGCAGTCACTTTCTGTCTAAAAAAACATGGGAGAATGATGGATGTGGAAAA NM_001314029.1 CCTATCATAGTCATTCAGTGATTG---TTGAATAAATGAATGAATGAATAACA------NR_052005.1 ------GGGAGAATGATGGATGTGAAAAA NM_014143.3 TGTAGCACTGACATTCATCTTCCG---TTTAAGAAAAGGGAGAATGATGGATGTGAAAAA NM_001267706.1 TGTAGCACTGACATTCATCTTCCG---TTTAAGAAAAGGGAGAATGATGGATGTGAAAAA * ****** *

AB898678.1 ATGTTGCACCCGAGATAGGAACTCAAAGAAACGAAATGATATACAATTTGAAGAGACATA XM_005615932.3 ATGTTGCACCCGAGATAGGAACTCAAAGAAACGAAATGATATACAATTTGAAGAGACATA XM_005615933.3 ATGTTGCACCCGAGATAGGAACTCAAAGAAACGAAATGATATACAATTTGAAGAGACATA XM_022421251.1 ATGTTGCACCCGAGATAGGAACTCAAAGAAACGAAATGATATACAATTTGAAGAGACATA XM_022421407.1 ATGTTGCACCCGAGATAGGAACTCAAAGAAACGAAATGATATACAATTTGAAGAGACATA XM_022421286.1 ATGTTGCACCCGAGATAGGAACTCAAAGAAACGAAATGATATACAATTTGAAGAGACATA XM_022421319.1 ATGTTGCACCCGAGATAGGAACTCAAAGAAACGAAATGATATACAATTTGAAGAGACATA XM_005615931.3 ATGTTGCACCCGAGATAGGAACTCAAAGAAACGAAATGATATACAATTTGAAGAGACATA XM_005615934.3 ATGTTGCACCCGAGATAGGAACTCAAAGAAACGAAATGATATACAATTTGAAGAGACATA NM_001314029.1 ------NR_052005.1 ATGTGGCATCCAAGATACAAACTCAAAGAAGCAAAGTGATACACATTTGGAGGAGACGTA NM_014143.3 ATGTGGCATCCAAGATACAAACTCAAAGAAGCAAAGTGATACACATTTGGAGGAGACGTA NM_001267706.1 ATGTGGCATCCAAGATACAAACTCAAAGAAGCAAAGTGATACACATTTGGAGGAGACGTA

AB898678.1 ATCCAGCATGGAAACTCCTGATCTTAAAGCAGGGATTCTCGGCCTGTGGTTTGAGTTCAG XM_005615932.3 ATCCAGCATGGAAACTCCTGATCTTAAAGCAGGGATTCTCGGCCTGTGGTTTGAGTTCAG XM_005615933.3 ATCCAGCATGGAAACTCCTGATCTTAAAGCAGGGATTCTCGGCCTGTGGTTTGAGTTCAG XM_022421251.1 ATCCAGCATGGAAACTCCTGATCTTAAAGCAGGGATTCTCGGCCTGTGGTTTGAGTTCAG XM_022421407.1 ATCCAGCATGGAAACTCCTGATCTTAAAGCAGGGATTCTCGGCCTGTGGTTTGAGTTCAG XM_022421286.1 ATCCAGCATGGAAACTCCTGATCTTAAAGCAGGGATTCTCGGCCTGTGGTTTGAGTTCAG XM_022421319.1 ATCCAGCATGGAAACTCCTGATCTTAAAGCAGGGATTCTCGGCCTGTGGTTTGAGTTCAG XM_005615931.3 ATCCAGCATGGAAACTCCTGATCTTAAAGCAGGGATTCTCGGCCTGTGGTTTGAGTTCAG XM_005615934.3 ATCCAGCATGGAAACTCCTGATCTTAAAGCAGGGATTCTCGGCCTGTGGTTTGAGTTCAG NM_001314029.1 ------NR_052005.1 ATCCAGCATTGGAACTTCTGATCTTCAAGCAGGGATTCTCAACCTGTGGTTTAGGGGTTC NM_014143.3 ATCCAGCATTGGAACTTCTGATCTTCAAGCAGGGATTCTCAACCTGTGGTTTAGGGGTTC NM_001267706.1 ATCCAGCATTGGAACTTCTGATCTTCAAGCAGGGATTCTCAACCTGTGGTTTAGGGGTTC

AB898678.1 CAGAGCTACTCATGACCAGAGCAGTGCAGAGGGCCAGGCAAGCTGCAGGCAACATAGGGC XM_005615932.3 CAGAGCTACTCATGACCAGAGCAGTGCAGAGGGCCAGGCAAGCTGCAGGCAACATAGGGC XM_005615933.3 CAGAGCTACTCATGACCAGAGCAGTGCAGAGGGCCAGGCAAGCTGCAGGCAACATAGGGC XM_022421251.1 CAGAGCTACTCATGACCAGAGCAGTGCAGAGGGCCAGGCAAGCTGCAGGCAACATAGGGC XM_022421407.1 CAGAGCTACTCATGACCAGAGCAGTGCAGAGGGCCAGGCAAGCTGCAGGCAACATAGGGC XM_022421286.1 CAGAGCTACTCATGACCAGAGCAGTGCAGAGGGCCAGGCAAGCTGCAGGCAACATAGGGC XM_022421319.1 CAGAGCTACTCATGACCAGAGCAGTGCAGAGGGCCAGGCAAGCTGCAGGCAACATAGGGC XM_005615931.3 CAGAGCTACTCATGACCAGAGCAGTGCAGAGGGCCAGGCAAGCTGCAGGCAACATAGGGC XM_005615934.3 CAGAGCTACTCATGACCAGAGCAGTGCAGAGGGCCAGGCAAGCTGCAGGCAACATAGGGC NM_001314029.1 ------NR_052005.1 ATCGGGGCTGAGCGTGACAAGAGGAAGGAATGGGCCCGTGGGATGCAGGCAATGTGGGAC NM_014143.3 ATCGGGGCTGAGCGTGACAAGAGGAAGGAATGGGCCCGTGGGATGCAGGCAATGTGGGAC NM_001267706.1 ATCGGGGCTGAGCGTGACAAGAGGAAGGAATGGGCCCGTGGGATGCAGGCAATGTGGGAC

AB898678.1 TCAGAAGGCCCAAGTGGTGACCAGTGGAAGGTGGGAGAGAAGAGGAGGAGAACAAAGTAA XM_005615932.3 TCAGAAGGCCCAAGTGGTGACCAGTGGAAGGTGGGAGAGAAGAGGAGGAGAACAAAGTAA

Appendices

XM_005615933.3 TCAGAAGGCCCAAGTGGTGACCAGTGGAAGGTGGGAGAGAAGAGGAGGAGAACAAAGTAA XM_022421251.1 TCAGAAGGCCCAAGTGGTGACCAGTGGAAGGTGGGAGAGAAGAGGAGGAGAACAAAGTAA XM_022421407.1 TCAGAAGGCCCAAGTGGTGACCAGTGGAAGGTGGGAGAGAAGAGGAGGAGAACAAAGTAA XM_022421286.1 TCAGAAGGCCCAAGTGGTGACCAGTGGAAGGTGGGAGAGAAGAGGAGGAGAACAAAGTAA XM_022421319.1 TCAGAAGGCCCAAGTGGTGACCAGTGGAAGGTGGGAGAGAAGAGGAGGAGAACAAAGTAA XM_005615931.3 TCAGAAGGCCCAAGTGGTGACCAGTGGAAGGTGGGAGAGAAGAGGAGGAGAACAAAGTAA XM_005615934.3 TCAGAAGGCCCAAGTGGTGACCAGTGGAAGGTGGGAGAGAAGAGGAGGAGAACAAAGTAA NM_001314029.1 ------NR_052005.1 TTAAAAGGCCCAAGCACTGAAAATGGAACCTGGCGAAAGCAGAGGAGGAGAATGAAGAAA NM_014143.3 TTAAAAGGCCCAAGCACTGAAAATGGAACCTGGCGAAAGCAGAGGAGGAGAATGAAGAAA NM_001267706.1 TTAAAAGGCCCAAGCACTGAAAATGGAACCTGGCGAAAGCAGAGGAGGAGAATGAAGAAA

AB898678.1 TAAAGAGTAGAGGAGGTAGCCTGCAAGGAGACTTTGGCACTTCAAAACGACTGGGAGAAT XM_005615932.3 TAAAGAGTAGAGGAGGTAGCCTGCAAGGAGACTTTGGCACTTCAAAACGACTGGGAGAAT XM_005615933.3 TAAAGAGTAGAGGAGGTAGCCTGCAAGGAGACTTTGGCACTTCAAAACGACTGGGAGAAT XM_022421251.1 TAAAGAGTAGAGGAGGTAGCCTGCAAGGAGACTTTGGCACTTCAAAACGACTGGGAGAAT XM_022421407.1 TAAAGAGTAGAGGAGGTAGCCTGCAAGGAGACTTTGGCACTTCAAAACGACTGGGAGAAT XM_022421286.1 TAAAGAGTAGAGGAGGTAGCCTGCAAGGAGACTTTGGCACTTCAAAACGACTGGGAGAAT XM_022421319.1 TAAAGAGTAGAGGAGGTAGCCTGCAAGGAGACTTTGGCACTTCAAAACGACTGGGAGAAT XM_005615931.3 TAAAGAGTAGAGGAGGTAGCCTGCAAGGAGACTTTGGCACTTCAAAACGACTGGGAGAAT XM_005615934.3 TAAAGAGTAGAGGAGGTAGCCTGCAAGGAGACTTTGGCACTTCAAAACGACTGGGAGAAT NM_001314029.1 ------NR_052005.1 GATGGAGTCAAACAGGGAGCCTGGAGGGAGACCTTGATACTTTCAAATGCCTGAGGGGCT NM_014143.3 GATGGAGTCAAACAGGGAGCCTGGAGGGAGACCTTGATACTTTCAAATGCCTGAGGGGCT NM_001267706.1 GATGGAGTCAAACAGGGAGCCTGGAGGGAGACCTTGATACTTTCAAATGCCTGAGGGGCT

AB898678.1 CACAGGCACCTACAAAAGAAAGGAAGGACACTTCTGGAAGAGGAACCTCCCCATGAACCG XM_005615932.3 CACAGGCACCTACAAAAGAAAGGAAGGACACTTCTGGAAGAGGAACCTCCCCATGAACCG XM_005615933.3 CACAGGCACCTACAAAAGAAAGGAAGGACACTTCTGGAAGAGGAACCTCCCCATGAACCG XM_022421251.1 CACAGGCACCTACAAAAGAAAGGAAGGACACTTCTGGAAGAGGAACCTCCCCATGAACCG XM_022421407.1 CACAGGCACCTACAAAAGAAAGGAAGGACACTTCTGGAAGAGGAACCTCCCCATGAACCG XM_022421286.1 CACAGGCACCTACAAAAGAAAGGAAGGACACTTCTGGAAGAGGAACCTCCCCATGAACCG XM_022421319.1 CACAGGCACCTACAAAAGAAAGGAAGGACACTTCTGGAAGAGGAACCTCCCCATGAACCG XM_005615931.3 CACAGGCACCTACAAAAGAAAGGAAGGACACTTCTGGAAGAGGAACCTCCCCATGAACCG XM_005615934.3 CACAGGCACCTACAAAAGAAAGGAAGGACACTTCTGGAAGAGGAACCTCCCCATGAACCG NM_001314029.1 ------NR_052005.1 CATCGACGCCTGTGACAGGGAGAAAGGATACTTCTGAACAAGGAGCCTCCAAGCAAATCA NM_014143.3 CATCGACGCCTGTGACAGGGAGAAAGGATACTTCTGAACAAGGAGCCTCCAAGCAAATCA NM_001267706.1 CATCGACGCCTGTGACAGGGAGAAAGGATACTTCTGAACAAGGAGCCTCCAAGCAAATCA

AB898678.1 TCCATCGCTCATCCTAGGAAAACAAGTTGCGATTCCCTGATTTAATGTGTCAGTTCCTGC XM_005615932.3 TCCATCGCTCATCCTAGGAAAACAAGTTGCGATTCCCTGATTTAATGTGTCAGTTCCTGC XM_005615933.3 TCCATCGCTCATCCTAGGAAAACAAGTTGCGATTCCCTGATTTAATGTGTCAGTTCCTGC XM_022421251.1 TCCATCGCTCATCCTAGGAAAACAAGTTGCGATTCCCTGATTTAATGTGTCAGTTCCTGC XM_022421407.1 TCCATCGCTCATCCTAGGAAAACAAGTTGCGATTCCCTGATTTAATGTGTCAGTTCCTGC XM_022421286.1 TCCATCGCTCATCCTAGGAAAACAAGTTGCGATTCCCTGATTTAATGTGTCAGTTCCTGC XM_022421319.1 TCCATCGCTCATCCTAGGAAAACAAGTTGCGATTCCCTGATTTAATGTGTCAGTTCCTGC XM_005615931.3 TCCATCGCTCATCCTAGGAAAACAAGTTGCGATTCCCTGATTTAATGTGTCAGTTCCTGC XM_005615934.3 TCCATCGCTCATCCTAGGAAAACAAGTTGCGATTCCCTGATTTAATGTGTCAGTTCCTGC NM_001314029.1 ------NR_052005.1 TCCATTGCTCATCCTAGGAAGACGGGTTGAGAATCCCTAATTT-GAGGGTCAGTTCCTGC NM_014143.3 TCCATTGCTCATCCTAGGAAGACGGGTTGAGAATCCCTAATTT-GAGGGTCAGTTCCTGC NM_001267706.1 TCCATTGCTCATCCTAGGAAGACGGGTTGAGAATCCCTAATTT-GAGGGTCAGTTCCTGC

AB898678.1 AGAAGTGCACTTTGCCTTCGCTCAACGTCTCTATTTGTCATCTGTGTGACTGAAGGTCCC

100

Appendices

XM_005615932.3 AGAAGTGCACTTTGCCTTCGCTCAACGTCTCTATTTGTCATCTGTGTGACTGAAGGTCCC XM_005615933.3 AGAAGTGCACTTTGCCTTCGCTCAACGTCTCTATTTGTCATCTGTGTGACTGAAGGTCCC XM_022421251.1 AGAAGTGCACTTTGCCTTCGCTCAACGTCTCTATTTGTCATCTGTGTGACTGAAGGTCCC XM_022421407.1 AGAAGTGCACTTTGCCTTCGCTCAACGTCTCTATTTGTCATCTGTGTGACTGAAGGTCCC XM_022421286.1 AGAAGTGCACTTTGCCTTCGCTCAACGTCTCTATTTGTCATCTGTGTGACTGAAGGTCCC XM_022421319.1 AGAAGTGCACTTTGCCTTCGCTCAACGTCTCTATTTGTCATCTGTGTGACTGAAGGTCCC XM_005615931.3 AGAAGTGCACTTTGCCTTCGCTCAACGTCTCTATTTGTCATCTGTGTGACTGAAGGTCCC XM_005615934.3 AGAAGTGCACTTTGCCTTCGCTCAACGTCTCTATTTGTCATCTGTGTGACTGAAGGTCCC NM_001314029.1 ------NR_052005.1 AGAAGTGCCCTTTGCCTCCACTCAATGCCTCAATTTGTTTTCTGCATGACTGAGAGTCTC NM_014143.3 AGAAGTGCCCTTTGCCTCCACTCAATGCCTCAATTTGTTTTCTGCATGACTGAGAGTCTC NM_001267706.1 AGAAGTGCCCTTTGCCTCCACTCAATGCCTCAATTTGTTTTCTGCATGACTGAGAGTCTC

AB898678.1 AGTGTTGCAACAGTATTTAAGGAT------GTTATTTCTATTTATTTTGAGTCTTT--- XM_005615932.3 AGTGTTGCAACAGTATTTAAGGAT------GTTATTTCTATTTATTTTGAGTCTTT--- XM_005615933.3 AGTGTTGCAACAGTATTTAAGGAT------GTTATTTCTATTTATTTTGAGTCTTT--- XM_022421251.1 AGTGTTGCAACAGTATTTAAGGAT------GTTATTTCTATTTATTTTGAGTCTTT--- XM_022421407.1 AGTGTTGCAACAGTATTTAAGGAT------GTTATTTCTATTTATTTTGAGTCTTT--- XM_022421286.1 AGTGTTGCAACAGTATTTAAGGAT------GTTATTTCTATTTATTTTGAGTCTTT--- XM_022421319.1 AGTGTTGCAACAGTATTTAAGGAT------GTTATTTCTATTTATTTTGAGTCTTT--- XM_005615931.3 AGTGTTGCAACAGTATTTAAGGAT------GTTATTTCTATTTATTTTGAGTCTTT--- XM_005615934.3 AGTGTTGCAACAGTATTTAAGGAT------GTTATTTCTATTTATTTTGAGTCTTT--- NM_001314029.1 ------NR_052005.1 AGTGTTGGAACGGGACAGTATTTATGTATGAGTTTTTCCTATTTATTTTGAGTCTGTGAG NM_014143.3 AGTGTTGGAACGGGACAGTATTTATGTATGAGTTTTTCCTATTTATTTTGAGTCTGTGAG NM_001267706.1 AGTGTTGGAACGGGACAGTATTTATGTATGAGTTTTTCCTATTTATTTTGAGTCTGTGAG

AB898678.1 GGAGTCTTGTCGTGTGAGTGTGGTTGTGAGTGATTTCTTTGGAAGACACATTGTAGTAGA XM_005615932.3 GGAGTCTTGTCGTGTGAGTGTGGTTGTGAGTGATTTCTTTGGAAGACACATTGTAGTAGA XM_005615933.3 GGAGTCTTGTCGTGTGAGTGTGGTTGTGAGTGATTTCTTTGGAAGACACATTGTAGTAGA XM_022421251.1 GGAGTCTTGTCGTGTGAGTGTGGTTGTGAGTGATTTCTTTGGAAGACACATTGTAGTAGA XM_022421407.1 GGAGTCTTGTCGTGTGAGTGTGGTTGTGAGTGATTTCTTTGGAAGACACATTGTAGTAGA XM_022421286.1 GGAGTCTTGTCGTGTGAGTGTGGTTGTGAGTGATTTCTTTGGAAGACACATTGTAGTAGA XM_022421319.1 GGAGTCTTGTCGTGTGAGTGTGGTTGTGAGTGATTTCTTTGGAAGACACATTGTAGTAGA XM_005615931.3 GGAGTCTTGTCGTGTGAGTGTGGTTGTGAGTGATTTCTTTGGAAGACACATTGTAGTAGA XM_005615934.3 GGAGTCTTGTCGTGTGAGTGTGGTTGTGAGTGATTTCTTTGGAAGACACATTGTAGTAGA NM_001314029.1 ------NR_052005.1 GTCTTCTTGTCATGTGAGTGTGGTTGTGAATGATTTCTTTTGAAGATATATTGTAGTAGA NM_014143.3 GTCTTCTTGTCATGTGAGTGTGGTTGTGAATGATTTCTTTTGAAGATATATTGTAGTAGA NM_001267706.1 GTCTTCTTGTCATGTGAGTGTGGTTGTGAATGATTTCTTTTGAAGATATATTGTAGTAGA

AB898678.1 AGTTAAAATTTTGTCACAAAACTACATTTACTGCTTAGTGAGTTGTGTGTGTTCAATAAA XM_005615932.3 AGTTAAAATTTTGTCACAAAACTACATTTACTGCTTAGTGAGTTGTGTGTGTTCAATAAA XM_005615933.3 AGTTAAAATTTTGTCACAAAACTACATTTACTGCTTAGTGAGTTGTGTGTGTTCAATAAA XM_022421251.1 AGTTAAAATTTTGTCACAAAACTACATTTACTGCTTAGTGAGTTGTGTGTGTTCAATAAA XM_022421407.1 AGTTAAAATTTTGTCACAAAACTACATTTACTGCTTAGTGAGTTGTGTGTGTTCAATAAA XM_022421286.1 AGTTAAAATTTTGTCACAAAACTACATTTACTGCTTAGTGAGTTGTGTGTGTTCAATAAA XM_022421319.1 AGTTAAAATTTTGTCACAAAACTACATTTACTGCTTAGTGAGTTGTGTGTGTTCAATAAA XM_005615931.3 AGTTAAAATTTTGTCACAAAACTACATTTACTGCTTAGTGAGTTGTGTGTGTTCAATAAA XM_005615934.3 AGTTAAAATTTTGTCACAAAACTACATTTACTGCTTAGTGAGTTGTGTGTGTTCAATAAA NM_001314029.1 ------NR_052005.1 TGTTACAATTTTGTCGCCAAACTAAACTTGCTGCTTAATGATTTGCTCACATCTAGTAAA NM_014143.3 TGTTACAATTTTGTCGCCAAACTAAACTTGCTGCTTAATGATTTGCTCACATCTAGTAAA NM_001267706.1 TGTTACAATTTTGTCGCCAAACTAAACTTGCTGCTTAATGATTTGCTCACATCTAGTAAA

AB898678.1 ACGTGTAGTATTAAAAAAAAAAAAAAA------XM_005615932.3 ACGTGTAGTATTTATAAGGTGTTTGGTATCCTCCACAGCCGCCAGGACATGGGAA--ACA

101

Appendices

XM_005615933.3 ACGTGTAGTATTTATAAGGTGTTTGGTATCCTCCACAGCCGCCAGGACATGGGAA--ACA XM_022421251.1 ACGTGTAGTATTTATAAGGTGTTTGGTATCCTCCACAGCCGCCAGGACATGGGAA--ACA XM_022421407.1 ACGTGTAGTATTTATAAGGTGTTTGGTATCCTCCACAGCCGCCAGGACATGGGAA--ACA XM_022421286.1 ACGTGTAGTATTTATAAGGTGTTTGGTATCCTCCACAGCCGCCAGGACATGGGAA--ACA XM_022421319.1 ACGTGTAGTATTTATAAGGTGTTTGGTATCCTCCACAGCCGCCAGGACATGGGAA--ACA XM_005615931.3 ACGTGTAGTATTTATAAGGTGTTTGGTATCCTCCACAGCCGCCAGGACATGGGAA--ACA XM_005615934.3 ACGTGTAGTATTTATAAGGTGTTTGGTATCCTCCACAGCCGCCAGGACATGGGAA--ACA NM_001314029.1 ------NR_052005.1 ACATGGAGTATTTGTAAGGTGCTTGGTCTCCTCTATAACTACAAGTATACATTGGAAGCA NM_014143.3 ACATGGAGTATTTGTAAGGTGCTTGGTCTCCTCTATAACTACAAGTATACATTGGAAGCA NM_001267706.1 ACATGGAGTATTTGTAAGGTGCTTGGTCTCCTCTATAACTACAAGTATACATTGGAAGCA

AB898678.1 ------XM_005615932.3 AGGAGAACAGACCCATGATTTCTATAGGATATTCGTTGCCTTATTTAACCCTCCAGTACT XM_005615933.3 AGGAGAACAGACCCATGATTTCTATAGGATATTCGTTGCCTTATTTAACCCTCCAGTACT XM_022421251.1 AGGAGAACAGACCCATGATTTCTATAGGATATTCGTTGCCTTATTTAACCCTCCAGTACT XM_022421407.1 AGGAGAACAGACCCATGATTTCTATAGGATATTCGTTGCCTTATTTAACCCTCCAGTACT XM_022421286.1 AGGAGAACAGACCCATGATTTCTATAGGATATTCGTTGCCTTATTTAACCCTCCAGTACT XM_022421319.1 AGGAGAACAGACCCATGATTTCTATAGGATATTCGTTGCCTTATTTAACCCTCCAGTACT XM_005615931.3 AGGAGAACAGACCCATGATTTCTATAGGATATTCGTTGCCTTATTTAACCCTCCAGTACT XM_005615934.3 AGGAGAACAGACCCATGATTTCTATAGGATATTCGTTGCCTTATTTAACCCTCCAGTACT NM_001314029.1 ------NR_052005.1 TAAAGATCAAACCGTTGGTTGC-AT---AGGATGTCACCTTTATTTAACCCATTAATACT NM_014143.3 TAAAGATCAAACCGTTGGTTGC-AT---AGGATGTCACCTTTATTTAACCCATTAATACT NM_001267706.1 TAAAGATCAAACCGTTGGTTGC-AT---AGGATGTCACCTTTATTTAACCCATTAATACT

AB898678.1 ------XM_005615932.3 TTGGTTGACCCTAATTATCTTCTCAGACCTGAAGGTAATATATGTTACTGCAGTATCTGT XM_005615933.3 TTGGTTGACCCTAATTATCTTCTCAGACCTGAAGGTAATATATGTTACTGCAGTATCTGT XM_022421251.1 TTGGTTGACCCTAATTATCTTCTCAGACCTGAAGGTAATATATGTTACTGCAGTATCTGT XM_022421407.1 TTGGTTGACCCTAATTATCTTCTCAGACCTGAAGGTAATATATGTTACTGCAGTATCTGT XM_022421286.1 TTGGTTGACCCTAATTATCTTCTCAGACCTGAAGGTAATATATGTTACTGCAGTATCTGT XM_022421319.1 TTGGTTGACCCTAATTATCTTCTCAGACCTGAAGGTAATATATGTTACTGCAGTATCTGT XM_005615931.3 TTGGTTGACCCTAATTATCTTCTCAGACCTGAAGGTAATATATGTTACTGCAGTATCTGT XM_005615934.3 TTGGTTGACCCTAATTATCTTCTCAGACCTGAAGGTAATATATGTTACTGCAGTATCTGT NM_001314029.1 ------NR_052005.1 CTGGTTGACC-TAATCTTATTCTCAGACCT------CAAGTGTCTGTGCAGTATCTGT NM_014143.3 CTGGTTGACC-TAATCTTATTCTCAGACCT------CAAGTGTCTGTGCAGTATCTGT NM_001267706.1 CTGGTTGACC-TAATCTTATTCTCAGACCT------CAAGTGTCTGTGCAGTATCTGT

AB898678.1 ------XM_005615932.3 TCCATTTTAAATATCAGTGTAAACCACTGTGTGGGATCCTACACAGAATCTCATTTAATG XM_005615933.3 TCCATTTTAAATATCAGTGTAAACCACTGTGTGGGATCCTACACAGAATCTCATTTAATG XM_022421251.1 TCCATTTTAAATATCAGTGTAAACCACTGTGTGGGATCCTACACAGAATCTCATTTAATG XM_022421407.1 TCCATTTTAAATATCAGTGTAAACCACTGTGTGGGATCCTACACAGAATCTCATTTAATG XM_022421286.1 TCCATTTTAAATATCAGTGTAAACCACTGTGTGGGATCCTACACAGAATCTCATTTAATG XM_022421319.1 TCCATTTTAAATATCAGTGTAAACCACTGTGTGGGATCCTACACAGAATCTCATTTAATG XM_005615931.3 TCCATTTTAAATATCAGTGTAAACCACTGTGTGGGATCCTACACAGAATCTCATTTAATG XM_005615934.3 TCCATTTTAAATATCAGTGTAAACCACTGTGTGGGATCCTACACAGAATCTCATTTAATG NM_001314029.1 ------NR_052005.1 TCCATTTAAATATCAGCTTTACAATTATGTGGTAGCCTACACACATAATCTCATTTCATC NM_014143.3 TCCATTTAAATATCAGCTTTACAATTATGTGGTAGCCTACACACATAATCTCATTTCATC NM_001267706.1 TCCATTTAAATATCAGCTTTACAATTATGTGGTAGCCTACACACATAATCTCATTTCATC

AB898678.1 ------XM_005615932.3 CCTAGGACAACTGTGTTGTGGTAGGAACTCTTACTGTACCATT-TCACAGTGGAGAGCGC XM_005615933.3 CCTAGGACAACTGTGTTGTGGTAGGAACTCTTACTGTACCATT-TCACAGTGGAGAGCGC

102

Appendices

XM_022421251.1 CCTAGGACAACTGTGTTGTGGTAGGAACTCTTACTGTACCATT-TCACAGTGGAGAGCGC XM_022421407.1 CCTAGGACAACTGTGTTGTGGTAGGAACTCTTACTGTACCATT-TCACAGTGGAGAGCGC XM_022421286.1 CCTAGGACAACTGTGTTGTGGTAGGAACTCTTACTGTACCATT-TCACAGTGGAGAGCGC XM_022421319.1 CCTAGGACAACTGTGTTGTGGTAGGAACTCTTACTGTACCATT-TCACAGTGGAGAGCGC XM_005615931.3 CCTAGGACAACTGTGTTGTGGTAGGAACTCTTACTGTACCATT-TCACAGTGGAGAGCGC XM_005615934.3 CCTAGGACAACTGTGTTGTGGTAGGAACTCTTACTGTACCATT-TCACAGTGGAGAGCGC NM_001314029.1 ------NR_052005.1 GCTGTAACCACCCTGTTGTGATAACCACTATTATTTTACCCATCGTACAGCTGAGGAAGC NM_014143.3 GCTGTAACCACCCTGTTGTGATAACCACTATTATTTTACCCATCGTACAGCTGAGGAAGC NM_001267706.1 GCTGTAACCACCCTGTTGTGATAACCACTATTATTTTACCCATCGTACAGCTGAGGAAGC

AB898678.1 ------XM_005615932.3 GAAGAGATCGAGTAACTTGCCTGAGAGAGTAAATAGCAGACCCTGGATGTCCACAACTGT XM_005615933.3 GAAGAGATCGAGTAACTTGCCTGAGAGAGTAAATAGCAGACCCTGGATGTCCACAACTGT XM_022421251.1 GAAGAGATCGAGTAACTTGCCTGAGAGAGTAAATAGCAGACCCTGGATGTCCACAACTGT XM_022421407.1 GAAGAGATCGAGTAACTTGCCTGAGAGAGTAAATAGCAGACCCTGGATGTCCACAACTGT XM_022421286.1 GAAGAGATCGAGTAACTTGCCTGAGAGAGTAAATAGCAGACCCTGGATGTCCACAACTGT XM_022421319.1 GAAGAGATCGAGTAACTTGCCTGAGAGAGTAAATAGCAGACCCTGGATGTCCACAACTGT XM_005615931.3 GAAGAGATCGAGTAACTTGCCTGAGAGAGTAAATAGCAGACCCTGGATGTCCACAACTGT XM_005615934.3 GAAGAGATCGAGTAACTTGCCTGAGAGAGTAAATAGCAGACCCTGGATGTCCACAACTGT NM_001314029.1 ------NR_052005.1 AAACAGATTAAGTAACTTGCCCAAACCAGTAAATAGCAGACCTCAGACTGCCACCC---- NM_014143.3 AAACAGATTAAGTAACTTGCCCAAACCAGTAAATAGCAGACCTCAGACTGCCACCC---- NM_001267706.1 AAACAGATTAAGTAACTTGCCCAAACCAGTAAATAGCAGACCTCAGACTGCCACCC----

AB898678.1 ------XM_005615932.3 ACCCCGCCCCCCGCCTTTAGAATACAGCCTAGAGCTGTCTTTTTTTTTTTTTAAAGATTT XM_005615933.3 ACCCCGCCCCCCGCCTTTAGAATACAGCCTAGAGCTGTCTTTTTTTTTTTTTAAAGATTT XM_022421251.1 ACCCCGCCCCCCGCCTTTAGAATACAGCCTAGAGCTGTCTTTTTTTTTTTTTAAAGATTT XM_022421407.1 ACCCCGCCCCCCGCCTTTAGAATACAGCCTAGAGCTGTCTTTTTTTTTTTTTAAAGATTT XM_022421286.1 ACCCCGCCCCCCGCCTTTAGAATACAGCCTAGAGCTGTCTTTTTTTTTTTTTAAAGATTT XM_022421319.1 ACCCCGCCCCCCGCCTTTAGAATACAGCCTAGAGCTGTCTTTTTTTTTTTTTAAAGATTT XM_005615931.3 ACCCCGCCCCCCGCCTTTAGAATACAGCCTAGAGCTGTCTTTTTTTTTTTTTAAAGATTT XM_005615934.3 ACCCCGCCCCCCGCCTTTAGAATACAGCCTAGAGCTGTCTTTTTTTTTTTTTAAAGATTT NM_001314029.1 ------NR_052005.1 ------ACTGTCCTTTTATAATACAATTTACAGCTATATTTTACTTTAAGCA---ATTC NM_014143.3 ------ACTGTCCTTTTATAATACAATTTACAGCTATATTTTACTTTAAGCA---ATTC NM_001267706.1 ------ACTGTCCTTTTATAATACAATTTACAGCTATATTTTACTTTAAGCA---ATTC

AB898678.1 ------XM_005615932.3 ATTTATTTATTTATGATAGACATAGAGAGAGAGAGGCAGAGACACAGGAGGAGGGAGAAG XM_005615933.3 ATTTATTTATTTATGATAGACATAGAGAGAGAGAGGCAGAGACACAGGAGGAGGGAGAAG XM_022421251.1 ATTTATTTATTTATGATAGACATAGAGAGAGAGAGGCAGAGACACAGGAGGAGGGAGAAG XM_022421407.1 ATTTATTTATTTATGATAGACATAGAGAGAGAGAGGCAGAGACACAGGAGGAGGGAGAAG XM_022421286.1 ATTTATTTATTTATGATAGACATAGAGAGAGAGAGGCAGAGACACAGGAGGAGGGAGAAG XM_022421319.1 ATTTATTTATTTATGATAGACATAGAGAGAGAGAGGCAGAGACACAGGAGGAGGGAGAAG XM_005615931.3 ATTTATTTATTTATGATAGACATAGAGAGAGAGAGGCAGAGACACAGGAGGAGGGAGAAG XM_005615934.3 ATTTATTTATTTATGATAGACATAGAGAGAGAGAGGCAGAGACACAGGAGGAGGGAGAAG NM_001314029.1 ------NR_052005.1 TTTTATTCAAAAACCATTT----AT----TAAGTGCCC------TTGCAATAT NM_014143.3 TTTTATTCAAAAACCATTT----AT----TAAGTGCCC------TTGCAATAT NM_001267706.1 TTTTATTCAAAAACCATTT----AT----TAAGTGCCC------TTGCAATAT

AB898678.1 ------XM_005615932.3 CAGGCTCCATGCCGGGAGCCTGACGTGGGACTCGATCCCGGGACTCCAGGATCGCGCCCT

103

Appendices

XM_005615933.3 CAGGCTCCATGCCGGGAGCCTGACGTGGGACTCGATCCCGGGACTCCAGGATCGCGCCCT XM_022421251.1 CAGGCTCCATGCCGGGAGCCTGACGTGGGACTCGATCCCGGGACTCCAGGATCGCGCCCT XM_022421407.1 CAGGCTCCATGCCGGGAGCCTGACGTGGGACTCGATCCCGGGACTCCAGGATCGCGCCCT XM_022421286.1 CAGGCTCCATGCCGGGAGCCTGACGTGGGACTCGATCCCGGGACTCCAGGATCGCGCCCT XM_022421319.1 CAGGCTCCATGCCGGGAGCCTGACGTGGGACTCGATCCCGGGACTCCAGGATCGCGCCCT XM_005615931.3 CAGGCTCCATGCCGGGAGCCTGACGTGGGACTCGATCCCGGGACTCCAGGATCGCGCCCT XM_005615934.3 CAGGCTCCATGCCGGGAGCCTGACGTGGGACTCGATCCCGGGACTCCAGGATCGCGCCCT NM_001314029.1 ------NR_052005.1 CAATCGCTGTGCCA------GGCATTGAATC------TACAGA--- NM_014143.3 CAATCGCTGTGCCA------GGCATTGAATC------TACAGA--- NM_001267706.1 CAATCGCTGTGCCA------GGCATTGAATC------TACAGA---

AB898678.1 ------XM_005615932.3 GGGCCAAAGGCAGGCGCCAAACCGCTGAGCCACCCAGGGATCCCCTAGAGCTGTCTTTTA XM_005615933.3 GGGCCAAAGGCAGGCGCCAAACCGCTGAGCCACCCAGGGATCCCCTAGAGCTGTCTTTTA XM_022421251.1 GGGCCAAAGGCAGGCGCCAAACCGCTGAGCCACCCAGGGATCCCCTAGAGCTGTCTTTTA XM_022421407.1 GGGCCAAAGGCAGGCGCCAAACCGCTGAGCCACCCAGGGATCCCCTAGAGCTGTCTTTTA XM_022421286.1 GGGCCAAAGGCAGGCGCCAAACCGCTGAGCCACCCAGGGATCCCCTAGAGCTGTCTTTTA XM_022421319.1 GGGCCAAAGGCAGGCGCCAAACCGCTGAGCCACCCAGGGATCCCCTAGAGCTGTCTTTTA XM_005615931.3 GGGCCAAAGGCAGGCGCCAAACCGCTGAGCCACCCAGGGATCCCCTAGAGCTGTCTTTTA XM_005615934.3 GGGCCAAAGGCAGGCGCCAAACCGCTGAGCCACCCAGGGATCCCCTAGAGCTGTCTTTTA NM_001314029.1 ------NR_052005.1 ------NM_014143.3 ------NM_001267706.1 ------

AB898678.1 ------XM_005615932.3 AGAAATTCATTCATTCAGCAAACATTTAGGAAGCAGCCATGTCACTATGCCAGGTGGTGA XM_005615933.3 AGAAATTCATTCATTCAGCAAACATTTAGGAAGCAGCCATGTCACTATGCCAGGTGGTGA XM_022421251.1 AGAAATTCATTCATTCAGCAAACATTTAGGAAGCAGCCATGTCACTATGCCAGGTGGTGA XM_022421407.1 AGAAATTCATTCATTCAGCAAACATTTAGGAAGCAGCCATGTCACTATGCCAGGTGGTGA XM_022421286.1 AGAAATTCATTCATTCAGCAAACATTTAGGAAGCAGCCATGTCACTATGCCAGGTGGTGA XM_022421319.1 AGAAATTCATTCATTCAGCAAACATTTAGGAAGCAGCCATGTCACTATGCCAGGTGGTGA XM_005615931.3 AGAAATTCATTCATTCAGCAAACATTTAGGAAGCAGCCATGTCACTATGCCAGGTGGTGA XM_005615934.3 AGAAATTCATTCATTCAGCAAACATTTAGGAAGCAGCCATGTCACTATGCCAGGTGGTGA NM_001314029.1 ------NR_052005.1 ------T NM_014143.3 ------T NM_001267706.1 ------T

AB898678.1 ------XM_005615932.3 CTAAGCAAGACAAAGTACCTCTCTCCCCAGGAGCTCGCAGTATCATGGGGGAGACTGATA XM_005615933.3 CTAAGCAAGACAAAGTACCTCTCTCCCCAGGAGCTCGCAGTATCATGGGGGAGACTGATA XM_022421251.1 CTAAGCAAGACAAAGTACCTCTCTCCCCAGGAGCTCGCAGTATCATGGGGGAGACTGATA XM_022421407.1 CTAAGCAAGACAAAGTACCTCTCTCCCCAGGAGCTCGCAGTATCATGGGGGAGACTGATA XM_022421286.1 CTAAGCAAGACAAAGTACCTCTCTCCCCAGGAGCTCGCAGTATCATGGGGGAGACTGATA XM_022421319.1 CTAAGCAAGACAAAGTACCTCTCTCCCCAGGAGCTCGCAGTATCATGGGGGAGACTGATA XM_005615931.3 CTAAGCAAGACAAAGTACCTCTCTCCCCAGGAGCTCGCAGTATCATGGGGGAGACTGATA XM_005615934.3 CTAAGCAAGACAAAGTACCTCTCTCCCCAGGAGCTCGCAGTATCATGGGGGAGACTGATA NM_001314029.1 ------NR_052005.1 GTGAGCAAGACAAAGTACCTGTCC-TCAAGGAGCTCATAGTATA-ATGAGGAGATTAACA NM_014143.3 GTGAGCAAGACAAAGTACCTGTCC-TCAAGGAGCTCATAGTATA-ATGAGGAGATTAACA NM_001267706.1 GTGAGCAAGACAAAGTACCTGTCC-TCAAGGAGCTCATAGTATA-ATGAGGAGATTAACA

AB898678.1 ------XM_005615932.3 AGAGAATTCATCATTTCAGTATAGTGCAGTGTCATAGTATAAGGTGTTGGAGGAGAATGA

104

Appendices

XM_005615933.3 AGAGAATTCATCATTTCAGTATAGTGCAGTGTCATAGTATAAGGTGTTGGAGGAGAATGA XM_022421251.1 AGAGAATTCATCATTTCAGTATAGTGCAGTGTCATAGTATAAGGTGTTGGAGGAGAATGA XM_022421407.1 AGAGAATTCATCATTTCAGTATAGTGCAGTGTCATAGTATAAGGTGTTGGAGGAGAATGA XM_022421286.1 AGAGAATTCATCATTTCAGTATAGTGCAGTGTCATAGTATAAGGTGTTGGAGGAGAATGA XM_022421319.1 AGAGAATTCATCATTTCAGTATAGTGCAGTGTCATAGTATAAGGTGTTGGAGGAGAATGA XM_005615931.3 AGAGAATTCATCATTTCAGTATAGTGCAGTGTCATAGTATAAGGTGTTGGAGGAGAATGA XM_005615934.3 AGAGAATTCATCATTTCAGTATAGTGCAGTGTCATAGTATAAGGTGTTGGAGGAGAATGA NM_001314029.1 ------NR_052005.1 AGAAAATGTATTATTACAATTTAGTCCAGTGTCATAGCATAAGGATGATGCGAGGGGAAA NM_014143.3 AGAAAATGTATTATTACAATTTAGTCCAGTGTCATAGCATAAGGATGATGCGAGGGGAAA NM_001267706.1 AGAAAATGTATTATTACAATTTAGTCCAGTGTCATAGCATAAGGATGATGCGAGGGGAAA

AB898678.1 ------XM_005615932.3 TGGCATGAAGAGGAGAGTCTAGAAAGGCTCCCAAAGGGCGGGGGGCATACCCCCCAGGGC XM_005615933.3 TGGCATGAAGAGGAGAGTCTAGAAAGGCTCCCAAAGGGCGGGGGGCATACCCCCCAGGGC XM_022421251.1 TGGCATGAAGAGGAGAGTCTAGAAAGGCTCCCAAAGGGCGGGGGGCATACCCCCCAGGGC XM_022421407.1 TGGCATGAAGAGGAGAGTCTAGAAAGGCTCCCAAAGGGCGGGGGGCATACCCCCCAGGGC XM_022421286.1 TGGCATGAAGAGGAGAGTCTAGAAAGGCTCCCAAAGGGCGGGGGGCATACCCCCCAGGGC XM_022421319.1 TGGCATGAAGAGGAGAGTCTAGAAAGGCTCCCAAAGGGCGGGGGGCATACCCCCCAGGGC XM_005615931.3 TGGCATGAAGAGGAGAGTCTAGAAAGGCTCCCAAAGGGCGGGGGGCATACCCCCCAGGGC XM_005615934.3 TGGCATGAAGAGGAGAGTCTAGAAAGGCTCCCAAAGGGCGGGGGGCATACCCCCCAGGGC NM_001314029.1 ------NR_052005.1 ACC------CGAGCAGTGTTGCCAAGAGGAGG---AAATAGGC-CAATGTG NM_014143.3 ACC------CGAGCAGTGTTGCCAAGAGGAGG---AAATAGGC-CAATGTG NM_001267706.1 ACC------CGAGCAGTGTTGCCAAGAGGAGG---AAATAGGC-CAATGTG

AB898678.1 ------XM_005615932.3 GTCTCGTCAGTGTGTTCCTTGCAAGTCGAAAATACATTTTAATCAAAGTGATTTTAGTTT XM_005615933.3 GTCTCGTCAGTGTGTTCCTTGCAAGTCGAAAATACATTTTAATCAAAGTGATTTTAGTTT XM_022421251.1 GTCTCGTCAGTGTGTTCCTTGCAAGTCGAAAATACATTTTAATCAAAGTGATTTTAGTTT XM_022421407.1 GTCTCGTCAGTGTGTTCCTTGCAAGTCGAAAATACATTTTAATCAAAGTGATTTTAGTTT XM_022421286.1 GTCTCGTCAGTGTGTTCCTTGCAAGTCGAAAATACATTTTAATCAAAGTGATTTTAGTTT XM_022421319.1 GTCTCGTCAGTGTGTTCCTTGCAAGTCGAAAATACATTTTAATCAAAGTGATTTTAGTTT XM_005615931.3 GTCTCGTCAGTGTGTTCCTTGCAAGTCGAAAATACATTTTAATCAAAGTGATTTTAGTTT XM_005615934.3 GTCTCGTCAGTGTGTTCCTTGCAAGTCGAAAATACATTTTAATCAAAGTGATTTTAGTTT NM_001314029.1 ------NR_052005.1 GTCTGGGACGGTT-----GGATATACTTAAACATCTTAATAATCAGAGTAATTTTCATTT NM_014143.3 GTCTGGGACGGTT-----GGATATACTTAAACATCTTAATAATCAGAGTAATTTTCATTT NM_001267706.1 GTCTGGGACGGTT-----GGATATACTTAAACATCTTAATAATCAGAGTAATTTTCATTT

AB898678.1 ------XM_005615932.3 AGGAGATAAGTCAGTGTTTTTAAAAAATTGTGGAAAGTAGCTCTCGAATGCCTTTTCAAA XM_005615933.3 AGGAGATAAGTCAGTGTTTTTAAAAAATTGTGGAAAGTAGCTCTCGAATGCCTTTTCAAA XM_022421251.1 AGGAGATAAGTCAGTGTTTTTAAAAAATTGTGGAAAGTAGCTCTCGAATGCCTTTTCAAA XM_022421407.1 AGGAGATAAGTCAGTGTTTTTAAAAAATTGTGGAAAGTAGCTCTCGAATGCCTTTTCAAA XM_022421286.1 AGGAGATAAGTCAGTGTTTTTAAAAAATTGTGGAAAGTAGCTCTCGAATGCCTTTTCAAA XM_022421319.1 AGGAGATAAGTCAGTGTTTTTAAAAAATTGTGGAAAGTAGCTCTCGAATGCCTTTTCAAA XM_005615931.3 AGGAGATAAGTCAGTGTTTTTAAAAAATTGTGGAAAGTAGCTCTCGAATGCCTTTTCAAA XM_005615934.3 AGGAGATAAGTCAGTGTTTTTAAAAAATTGTGGAAAGTAGCTCTCGAATGCCTTTTCAAA NM_001314029.1 ------NR_052005.1 ACAAAGAGAGGTCGGTACTTAAAATAACCCTGAAAAATAACACTGGAATTCCTTTTCTAG NM_014143.3 ACAAAGAGAGGTCGGTACTTAAAATAACCCTGAAAAATAACACTGGAATTCCTTTTCTAG NM_001267706.1 ACAAAGAGAGGTCGGTACTTAAAATAACCCTGAAAAATAACACTGGAATTCCTTTTCTAG

AB898678.1 ------XM_005615932.3 ATATTATTATATTTACATCTGATTTATTTGCCTTTGCCATGCAATCTGACATTTATGTAT

105

Appendices

XM_005615933.3 ATATTATTATATTTACATCTGATTTATTTGCCTTTGCCATGCAATCT TTATGTAT XM_022421251.1 ATATTATTATATTTACATCTGATTTATTTGCCTTTGCCATGCAATCTGACATTTATGTAT XM_022421407.1 ATATTATTATATTTACATCTGATTTATTTGCCTTTGCCATGCAATCTGACATTTATGTAT XM_022421286.1 ATATTATTATATTTACATCTGATTTATTTGCCTTTGCCATGCAATCTGACATTTATGTAT XM_022421319.1 ATATTATTATATTTACATCTGATTTATTTGCCTTTGCCATGCAATCTGACATTTATGTAT XM_005615931.3 ATATTATTATATTTACATCTGATTTATTTGCCTTTGCCATGCAATCTGACATTTATGTAT XM_005615934.3 ATATTATTATATTTACATCTGATTTATTTGCCTTTGCCATGCAATCTGACATTTATGTAT NM_001314029.1 ------NR_052005.1 CATTA----T----ATTTATTCCTGATTTGCCTTTGCCATATAATCTAATGCTTGTTTAT NM_014143.3 CATTA----T----ATTTATTCCTGATTTGCCTTTGCCATATAATCTAATGCTTGTTTAT NM_001267706.1 CATTA----T----ATTTATTCCTGATTTGCCTTTGCCATATAATCTAATGCTTGTTTAT

AB898678.1 ------XM_005615932.3 ATGGTGTTGGTATTGCTCAGTGGTTCTTTCTTTTTTTTTATATATATAAGTGCAATTGAA XM_005615933.3 ATGGTGTTGGTATTGCTCAGTGGTTCTTTCTTTTTTTTTATATATATAAGTGCAATTGAA XM_022421251.1 ATGGTGTTGGTATTGCTCAGTGGTTCTTTCTTTTTTTTTATATATATAAGTGCAATTGAA XM_022421407.1 ATGGTGTTGGTATTGCTCAGTGGTTCTTTCTTTTTTTTTATATATATAAGTGCAATTGAA XM_022421286.1 ATGGTGTTGGTATTGCTCAGTGGTTCTTTCTTTTTTTTTATATATATAAGTGCAATTGAA XM_022421319.1 ATGGTGTTGGTATTGCTCAGTGGTTCTTTCTTTTTTTTTATATATATAAGTGCAATTGAA XM_005615931.3 ATGGTGTTGGTATTGCTCAGTGGTTCTTTCTTTTTTTTTATATATATAAGTGCAATTGAA XM_005615934.3 ATGGTGTTGGTATTGCTCAGTGGTTCTTTCTTTTTTTTTATATATATAAGTGCAATTGAA NM_001314029.1 ------NR_052005.1 ATAGTGTCTGG------TATTGTTTAACAGTTCTGTCTTTTCTATTTAAATGCCACTAAA NM_014143.3 ATAGTGTCTGG------TATTGTTTAACAGTTCTGTCTTTTCTATTTAAATGCCACTAAA NM_001267706.1 ATAGTGTCTGG------TATTGTTTAACAGTTCTGTCTTTTCTATTTAAATGCCACTAAA

AB898678.1 ------XM_005615932.3 GTATGAATTCATACCTTTCCATGATTCAAAATTCAGAAAGTCCAAGAGGAGACATTA-AA XM_005615933.3 GTATGAATTCATACCTTTCCATGATTCAAAATTCAGAAAGTCCAAGAGGAGACATTA-AA XM_022421251.1 GTATGAATTCATACCTTTCCATGATTCAAAATTCAGAAAGTCCAAGAGGAGACATTA-AA XM_022421407.1 GTATGAATTCATACCTTTCCATGATTCAAAATTCAGAAAGTCCAAGAGGAGACATTA-AA XM_022421286.1 GTATGAATTCATACCTTTCCATGATTCAAAATTCAGAAAGTCCAAGAGGAGACATTA-AA XM_022421319.1 GTATGAATTCATACCTTTCCATGATTCAAAATTCAGAAAGTCCAAGAGGAGACATTA-AA XM_005615931.3 GTATGAATTCATACCTTTCCATGATTCAAAATTCAGAAAGTCCAAGAGGAGACATTA-AA XM_005615934.3 GTATGAATTCATACCTTTCCATGATTCAAAATTCAGAAAGTCCAAGAGGAGACATTA-AA NM_001314029.1 ------NR_052005.1 TTTTAAATTCATACCTTTCCATGATTCAAAATTCAAAAGATCCCATGGGAGATGGTTGGA NM_014143.3 TTTTAAATTCATACCTTTCCATGATTCAAAATTCAAAAGATCCCATGGGAGATGGTTGGA NM_001267706.1 TTTTAAATTCATACCTTTCCATGATTCAAAATTCAAAAGATCCCATGGGAGATGGTTGGA

AB898678.1 ------XM_005615932.3 AAGTCCCCATTCCATCCTCCTAGATACCTGGTTTTTCTTTCCAGAAGCAACCAACTCTTG XM_005615933.3 AAGTCCCCATTCCATCCTCCTAGATACCTGGTTTTTCTTTCCAGAAGCAACCAACTCTTG XM_022421251.1 AAGTCCCCATTCCATCCTCCTAGATACCTGGTTTTTCTTTCCAGAAGCAACCAACTCTTG XM_022421407.1 AAGTCCCCATTCCATCCTCCTAGATACCTGGTTTTTCTTTCCAGAAGCAACCAACTCTTG XM_022421286.1 AAGTCCCCATTCCATCCTCCTAGATACCTGGTTTTTCTTTCCAGAAGCAACCAACTCTTG XM_022421319.1 AAGTCCCCATTCCATCCTCCTAGATACCTGGTTTTTCTTTCCAGAAGCAACCAACTCTTG XM_005615931.3 AAGTCCCCATTCCATCCTCCTAGATACCTGGTTTTTCTTTCCAGAAGCAACCAACTCTTG XM_005615934.3 AAGTCCCCATTCCATCCTCCTAGATACCTGGTTTTTCTTTCCAGAAGCAACCAACTCTTG NM_001314029.1 ------NR_052005.1 AAATCTCCACTTCATCCTCCAAGCCATTCAAGTTTCCTTTCCAGAAGCAA-CTGCTACTG NM_014143.3 AAATCTCCACTTCATCCTCCAAGCCATTCAAGTTTCCTTTCCAGAAGCAA-CTGCTACTG NM_001267706.1 AAATCTCCACTTCATCCTCCAAGCCATTCAAGTTTCCTTTCCAGAAGCAA-CTGCTACTG

AB898678.1 ------XM_005615932.3 CCTTTTGTTCTTATAGTCTTCTAAAGATATTCTAGGAATGTATGTAGAAATACATATAAA XM_005615933.3 CCTTTTGTTCTTATAGTCTTCTAAAGATATTCTAGGAATGTATGTAGAAATACATATAAA

106

Appendices

XM_022421251.1 CCTTTTGTTCTTATAGTCTTCTAAAGATATTCTAGGAATGTATGTAGAAATACATATAAA XM_022421407.1 CCTTTTGTTCTTATAGTCTTCTAAAGATATTCTAGGAATGTATGTAGAAATACATATAAA XM_022421286.1 CCTTTTGTTCTTATAGTCTTCTAAAGATATTCTAGGAATGTATGTAGAAATACATATAAA XM_022421319.1 CCTTTTGTTCTTATAGTCTTCTAAAGATATTCTAGGAATGTATGTAGAAATACATATAAA XM_005615931.3 CCTTTTGTTCTTATAGTCTTCTAAAGATATTCTAGGAATGTATGTAGAAATACATATAAA XM_005615934.3 CCTTTTGTTCTTATAGTCTTCTAAAGATATTCTAGGAATGTATGTAGAAATACATATAAA NM_001314029.1 ------NR_052005.1 CCTTTCATTCATATGTTCTTCTAAAGATAGTCTACATTTGGAAATGTATGTTAAA----- NM_014143.3 CCTTTCATTCATATGTTCTTCTAAAGATAGTCTACATTTGGAAATGTATGTTAAA----- NM_001267706.1 CCTTTCATTCATATGTTCTTCTAAAGATAGTCTACATTTGGAAATGTATGTTAAA-----

AB898678.1 ------XM_005615932.3 AATATATTTTTTATTTCTTCCTCTTTTTTGCATAAATGGTAACACACTATTCACCTGCTG XM_005615933.3 AATATATTTTTTATTTCTTCCTCTTTTTTGCATAAATGGTAACACACTATTCACCTGCTG XM_022421251.1 AATATATTTTTTATTTCTTCCTCTTTTTTGCATAAATGGTAACACACTATTCACCTGCTG XM_022421407.1 AATATATTTTTTATTTCTTCCTCTTTTTTGCATAAATGGTAACACACTATTCACCTGCTG XM_022421286.1 AATATATTTTTTATTTCTTCCTCTTTTTTGCATAAATGGTAACACACTATTCACCTGCTG XM_022421319.1 AATATATTTTTTATTTCTTCCTCTTTTTTGCATAAATGGTAACACACTATTCACCTGCTG XM_005615931.3 AATATATTTTTTATTTCTTCCTCTTTTTTGCATAAATGGTAACACACTATTCACCTGCTG XM_005615934.3 AATATATTTTTTATTTCTTCCTCTTTTTTGCATAAATGGTAACACACTATTCACCTGCTG NM_001314029.1 ------NR_052005.1 ------AGCACGTATTTTTAAAATTTTTTTCCTAAATAGTAACACATTGTATGTCTGCTG NM_014143.3 ------AGCACGTATTTTTAAAATTTTTTTCCTAAATAGTAACACATTGTATGTCTGCTG NM_001267706.1 ------AGCACGTATTTTTAAAATTTTTTTCCTAAATAGTAACACATTGTATGTCTGCTG

AB898678.1 ------XM_005615932.3 TGCACTTGGCTATTCTTATTTAATATATTACTTCTTATGGTGCCGTTGGAACAAATGTGA XM_005615933.3 TGCACTTGGCTATTCTTATTTAATATATTACTTCTTATGGTGCCGTTGGAACAAATGTGA XM_022421251.1 TGCACTTGGCTATTCTTATTTAATATATTACTTCTTATGGTGCCGTTGGAACAAATGTGA XM_022421407.1 TGCACTTGGCTATTCTTATTTAATATATTACTTCTTATGGTGCCGTTGGAACAAATGTGA XM_022421286.1 TGCACTTGGCTATTCTTATTTAATATATTACTTCTTATGGTGCCGTTGGAACAAATGTGA XM_022421319.1 TGCACTTGGCTATTCTTATTTAATATATTACTTCTTATGGTGCCGTTGGAACAAATGTGA XM_005615931.3 TGCACTTGGCTATTCTTATTTAATATATTACTTCTTATGGTGCCGTTGGAACAAATGTGA XM_005615934.3 TGCACTTGGCTATTCTTATTTAATATATTACTTCTTATGGTGCCGTTGGAACAAATGTGA NM_001314029.1 ------NR_052005.1 TGTACTTTGCTATTTTTATTTATTTTAGTGTTTCTTATATAGCAGATGGAATGAATTTGA NM_014143.3 TGTACTTTGCTATTTTTATTTATTTTAGTGTTTCTTATATAGCAGATGGAATGAATTTGA NM_001267706.1 TGTACTTTGCTATTTTTATTTATTTTAGTGTTTCTTATATAGCAGATGGAATGAATTTGA

AB898678.1 ------XM_005615932.3 AGTTCCCAGAAGTCAGGCACTGGGTCTTATTTATTTCTAGATTAGCTTTTCCATAGCCTG XM_005615933.3 AGTTCCCAGAAGTCAGGCACTGGGTCTTATTTATTTCTAGATTAGCTTTTCCATAGCCTG XM_022421251.1 AGTTCCCAGAAGTCAGGCACTGGGTCTTATTTATTTCTAGATTAGCTTTTCCATAGCCTG XM_022421407.1 AGTTCCCAGAAGTCAGGCACTGGGTCTTATTTATTTCTAGATTAGCTTTTCCATAGCCTG XM_022421286.1 AGTTCCCAGAAGTCAGGCACTGGGTCTTATTTATTTCTAGATTAGCTTTTCCATAGCCTG XM_022421319.1 AGTTCCCAGAAGTCAGGCACTGGGTCTTATTTATTTCTAGATTAGCTTTTCCATAGCCTG XM_005615931.3 AGTTCCCAGAAGTCAGGCACTGGGTCTTATTTATTTCTAGATTAGCTTTTCCATAGCCTG XM_005615934.3 AGTTCCCAGAAGTCAGGCACTGGGTCTTATTTATTTCTAGATTAGCTTTTCCATAGCCTG NM_001314029.1 ------NR_052005.1 AGTTCCCAGGGCTGAGGATCCATGCCTTCTTTGTTTCTAAGTTATCTTTCCCATAGCTTT NM_014143.3 AGTTCCCAGGGCTGAGGATCCATGCCTTCTTTGTTTCTAAGTTATCTTTCCCATAGCTTT NM_001267706.1 AGTTCCCAGGGCTGAGGATCCATGCCTTCTTTGTTTCTAAGTTATCTTTCCCATAGCTTT

AB898678.1 ------XM_005615932.3 TTATTACTT-TTATAAGACTTACTACATACTAAGTATGTGCTTCATTGACATTTGAACAA XM_005615933.3 TTATTACTT-TTATAAGACTTACTACATACTAAGTATGTGCTTCATTGACATTTGAACAA XM_022421251.1 TTATTACTT-TTATAAGACTTACTACATACTAAGTATGTGCTTCATTGACATTTGAACAA

107

Appendices

XM_022421407.1 TTATTACTT-TTATAAGACTTACTACATACTAAGTATGTGCTTCATTGACATTTGAACAA XM_022421286.1 TTATTACTT-TTATAAGACTTACTACATACTAAGTATGTGCTTCATTGACATTTGAACAA XM_022421319.1 TTATTACTT-TTATAAGACTTACTACATACTAAGTATGTGCTTCATTGACATTTGAACAA XM_005615931.3 TTATTACTT-TTATAAGACTTACTACATACTAAGTATGTGCTTCATTGACATTTGAACAA XM_005615934.3 TTATTACTT-TTATAAGACTTACTACATACTAAGTATGTGCTTCATTGACATTTGAACAA NM_001314029.1 ------NR_052005.1 TCATTATCTTTCATATGATCCAGTATATGTTAAATATGTCCTACATATACATTTAGACAA NM_014143.3 TCATTATCTTTCATATGATCCAGTATATGTTAAATATGTCCTACATATACATTTAGACAA NM_001267706.1 TCATTATCTTTCATATGATCCAGTATATGTTAAATATGTCCTACATATACATTTAGACAA

AB898678.1 ------XM_005615932.3 CCAGCATTTGTTGAGTGCTTGCTCCATGACTGAGTTTGGATTTGGTTTATTTTGTGTTTC XM_005615933.3 CCAGCATTTGTTGAGTGCTTGCTCCATGACTGAGTTTGGATTTGGTTTATTTTGTGTTTC XM_022421251.1 CCAGCATTTGTTGAGTGCTTGCTCCATGACTGAGTTTGGATTTGGTTTATTTTGTGTTTC XM_022421407.1 CCAGCATTTGTTGAGTGCTTGCTCCATGACTGAGTTTGGATTTGGTTTATTTTGTGTTTC XM_022421286.1 CCAGCATTTGTTGAGTGCTTGCTCCATGACTGAGTTTGGATTTGGTTTATTTTGTGTTTC XM_022421319.1 CCAGCATTTGTTGAGTGCTTGCTCCATGACTGAGTTTGGATTTGGTTTATTTTGTGTTTC XM_005615931.3 CCAGCATTTGTTGAGTGCTTGCTCCATGACTGAGTTTGGATTTGGTTTATTTTGTGTTTC XM_005615934.3 CCAGCATTTGTTGAGTGCTTGCTCCATGACTGAGTTTGGATTTGGTTTATTTTGTGTTTC NM_001314029.1 ------NR_052005.1 CCACCATTTGTTAAGTATTTGCTCTAGGACAGAGTTTGGATTTGTTTAT-----GTTTGC NM_014143.3 CCACCATTTGTTAAGTATTTGCTCTAGGACAGAGTTTGGATTTGTTTAT-----GTTTGC NM_001267706.1 CCACCATTTGTTAAGTATTTGCTCTAGGACAGAGTTTGGATTTGTTTAT-----GTTTGC

AB898678.1 ------XM_005615932.3 TCAAAGTGAGACACTTGGGTTCTCCAGGGTACACTGGCTCAACCTAGTCCTAACGACTG- XM_005615933.3 TCAAAGTGAGACACTTGGGTTCTCCAGGGTACACTGGCTCAACCTAGTCCTAACGACTG- XM_022421251.1 TCAAAGTGAGACACTTGGGTTCTCCAGGGTACACTGGCTCAACCTAGTCCTAACGACTG- XM_022421407.1 TCAAAGTGAGACACTTGGGTTCTCCAGGGTACACTGGCTCAACCTAGTCCTAACGACTG- XM_022421286.1 TCAAAGTGAGACACTTGGGTTCTCCAGGGTACACTGGCTCAACCTAGTCCTAACGACTG- XM_022421319.1 TCAAAGTGAGACACTTGGGTTCTCCAGGGTACACTGGCTCAACCTAGTCCTAACGACTG- XM_005615931.3 TCAAAGTGAGACACTTGGGTTCTCCAGGGTACACTGGCTCAACCTAGTCCTAACGACTG- XM_005615934.3 TCAAAGTGAGACACTTGGGTTCTCCAGGGTACACTGGCTCAACCTAGTCCTAACGACTG- NM_001314029.1 ------NR_052005.1 TCAAAAGGAGACCCATGGGCTCTCCAGGGTGCACTGAGTCAATCTAGTCCTAAAAAGCAA NM_014143.3 TCAAAAGGAGACCCATGGGCTCTCCAGGGTGCACTGAGTCAATCTAGTCCTAAAAAGCAA NM_001267706.1 TCAAAAGGAGACCCATGGGCTCTCCAGGGTGCACTGAGTCAATCTAGTCCTAAAAAGCAA

AB898678.1 ------XM_005615932.3 ACTTATTACCAATTCTGTTCGACAGAATCCTGTATGGAACTTGTGTTTTTTTTCCTATCT XM_005615933.3 ACTTATTACCAATTCTGTTCGACAGAATCCTGTATGGAACTTGTGTTTTTTTTCCTATCT XM_022421251.1 ACTTATTACCAATTCTGTTCGACAGAATCCTGTATGGAACTTGTGTTTTTTTTCCTATCT XM_022421407.1 ACTTATTACCAATTCTGTTCGACAGAATCCTGTATGGAACTTGTGTTTTTTTTCCTATCT XM_022421286.1 ACTTATTACCAATTCTGTTCGACAGAATCCTGTATGGAACTTGTGTTTTTTTTCCTATCT XM_022421319.1 ACTTATTACCAATTCTGTTCGACAGAATCCTGTATGGAACTTGTGTTTTTTTTCCTATCT XM_005615931.3 ACTTATTACCAATTCTGTTCGACAGAATCCTGTATGGAACTTGTGTTTTTTTTCCTATCT XM_005615934.3 ACTTATTACCAATTCTGTTCGACAGAATCCTGTATGGAACTTGTGTTTTTTTTCCTATCT NM_001314029.1 ------NR_052005.1 TCTTATTATTAACTCTGTATGACAGAATCATGTCTGGAACTTTTGTTTTCTG------NM_014143.3 TCTTATTATTAACTCTGTATGACAGAATCATGTCTGGAACTTTTGTTTTCTG------NM_001267706.1 TCTTATTATTAACTCTGTATGACAGAATCATGTCTGGAACTTTTGTTTTCTG------

108

Appendices

AB898678.1 ------XM_005615932.3 GTTTTCTCTTGAGTGAAAACTAGATTTTGATATTGCAGTTGTGAAATTACATGGGTGTTT XM_005615933.3 GTTTTCTCTTGAGTGAAAACTAGATTTTGATATTGCAGTTGTGAAATTACATGGGTGTTT XM_022421251.1 GTTTTCTCTTGAGTGAAAACTAGATTTTGATATTGCAGTTGTGAAATTACATGGGTGTTT XM_022421407.1 GTTTTCTCTTGAGTGAAAACTAGATTTTGATATTGCAGTTGTGAAATTACATGGGTGTTT XM_022421286.1 GTTTTCTCTTGAGTGAAAACTAGATTTTGATATTGCAGTTGTGAAATTACATGGGTGTTT XM_022421319.1 GTTTTCTCTTGAGTGAAAACTAGATTTTGATATTGCAGTTGTGAAATTACATGGGTGTTT XM_005615931.3 GTTTTCTCTTGAGTGAAAACTAGATTTTGATATTGCAGTTGTGAAATTACATGGGTGTTT XM_005615934.3 GTTTTCTCTTGAGTGAAAACTAGATTTTGATATTGCAGTTGTGAAATTACATGGGTGTTT NM_001314029.1 ------NR_052005.1 -CTTTCTGTCAAGTATAAACTTCACTTTGATGCTGTACTTGCAAAATCACATT------NM_014143.3 -CTTTCTGTCAAGTATAAACTTCACTTTGATGCTGTACTTGCAAAATCACATT------NM_001267706.1 -CTTTCTGTCAAGTATAAACTTCACTTTGATGCTGTACTTGCAAAATCACATT------

AB898678.1 ------XM_005615932.3 TTTTGTTTGTTTGTTTTTGGAAATTCCAGTAGTGTACCTTG-----ACTCTCACCTGTGC XM_005615933.3 TTTTGTTTGTTTGTTTTTGGAAATTCCAGTAGTGTACCTTG-----ACTCTCACCTGTGC XM_022421251.1 TTTTGTTTGTTTGTTTTTGGAAATTCCAGTAGTGTACCTTG-----ACTCTCACCTGTGC XM_022421407.1 TTTTGTTTGTTTGTTTTTGGAAATTCCAGTAGTGTACCTTG-----ACTCTCACCTGTGC XM_022421286.1 TTTTGTTTGTTTGTTTTTGGAAATTCCAGTAGTGTACCTTG-----ACTCTCACCTGTGC XM_022421319.1 TTTTGTTTGTTTGTTTTTGGAAATTCCAGTAGTGTACCTTG-----ACTCTCACCTGTGC XM_005615931.3 TTTTGTTTGTTTGTTTTTGGAAATTCCAGTAGTGTACCTTG-----ACTCTCACCTGTGC XM_005615934.3 TTTTGTTTGTTTGTTTTTGGAAATTCCAGTAGTGTACCTTG-----ACTCTCACCTGTGC NM_001314029.1 ------NR_052005.1 ------TTCTTTCTGGAAATTCCGGCAGTGTACCTTGACTGCTAGCTACCCTGTGC NM_014143.3 ------TTCTTTCTGGAAATTCCGGCAGTGTACCTTGACTGCTAGCTACCCTGTGC NM_001267706.1 ------TTCTTTCTGGAAATTCCGGCAGTGTACCTTGACTGCTAGCTACCCTGTGC

AB898678.1 ------XM_005615932.3 TTAAAAAAGGTCATGAGCTGTGCCTGAACCTCTGAGTCCCACCAGTTCTCATCACTACAT XM_005615933.3 TTAAAAAAGGTCATGAGCTGTGCCTGAACCTCTGAGTCCCACCAGTTCTCATCACTACAT XM_022421251.1 TTAAAAAAGGTCATGAGCTGTGCCTGAACCTCTGAGTCCCACCAGTTCTCATCACTACAT XM_022421407.1 TTAAAAAAGGTCATGAGCTGTGCCTGAACCTCTGAGTCCCACCAGTTCTCATCACTACAT XM_022421286.1 TTAAAAAAGGTCATGAGCTGTGCCTGAACCTCTGAGTCCCACCAGTTCTCATCACTACAT XM_022421319.1 TTAAAAAAGGTCATGAGCTGTGCCTGAACCTCTGAGTCCCACCAGTTCTCATCACTACAT XM_005615931.3 TTAAAAAAGGTCATGAGCTGTGCCTGAACCTCTGAGTCCCACCAGTTCTCATCACTACAT XM_005615934.3 TTAAAAAAGGTCATGAGCTGTGCCTGAACCTCTGAGTCCCACCAGTTCTCATCACTACAT NM_001314029.1 ------NR_052005.1 CAGAAAAGCCTCATTCGTTGTGCTTGAACCCTTGAATGCCACCAGCTGTCATCACTACAC NM_014143.3 CAGAAAAGCCTCATTCGTTGTGCTTGAACCCTTGAATGCCACCAGCTGTCATCACTACAC NM_001267706.1 CAGAAAAGCCTCATTCGTTGTGCTTGAACCCTTGAATGCCACCAGCTGTCATCACTACAC

AB898678.1 ------XM_005615932.3 AGATCTAAGACGTTGCTTTTCAAAGGCTTTGAGGTTCATGTGCCTCCCAGGAGGTCCTGT XM_005615933.3 AGATCTAAGACGTTGCTTTTCAAAGGCTTTGAGGTTCATGTGCCTCCCAGGAGGTCCTGT XM_022421251.1 AGATCTAAGACGTTGCTTTTCAAAGGCTTTGAGGTTCATGTGCCTCCCAGGAGGTCCTGT XM_022421407.1 AGATCTAAGACGTTGCTTTTCAAAGGCTTTGAGGTTCATGTGCCTCCCAGGAGGTCCTGT XM_022421286.1 AGATCTAAGACGTTGCTTTTCAAAGGCTTTGAGGTTCATGTGCCTCCCAGGAGGTCCTGT XM_022421319.1 AGATCTAAGACGTTGCTTTTCAAAGGCTTTGAGGTTCATGTGCCTCCCAGGAGGTCCTGT XM_005615931.3 AGATCTAAGACGTTGCTTTTCAAAGGCTTTGAGGTTCATGTGCCTCCCAGGAGGTCCTGT XM_005615934.3 AGATCTAAGACGTTGCTTTTCAAAGGCTTTGAGGTTCATGTGCCTCCCAGGAGGTCCTGT NM_001314029.1 ------NR_052005.1 AGCCCTCCTAAGAGGCTTCCTGGAGGTTTCGAGATTCAGATGCCCTG-GGAGATCCCAGA NM_014143.3 AGCCCTCCTAAGAGGCTTCCTGGAGGTTTCGAGATTCAGATGCCCTG-GGAGATCCCAGA NM_001267706.1 AGCCCTCCTAAGAGGCTTCCTGGAGGTTTCGAGATTCAGATGCCCTG-GGAGATCCCAGA

109

Appendices

AB898678.1 ------XM_005615932.3 GTCTTCCTTCTCTCTTGGCCATTTACCTGGATTATCGACAAAGAAAACCTTCATTTTGCC XM_005615933.3 GTCTTCCTTCTCTCTTGGCCATTTACCTGGATTATCGACAAAGAAAACCTTCATTTTGCC XM_022421251.1 GTCTTCCTTCTCTCTTGGCCATTTACCTGGATTATCGACAAAGAAAACCTTCATTTTGCC XM_022421407.1 GTCTTCCTTCTCTCTTGGCCATTTACCTGGATTATCGACAAAGAAAACCTTCATTTTGCC XM_022421286.1 GTCTTCCTTCTCTCTTGGCCATTTACCTGGATTATCGACAAAGAAAACCTTCATTTTGCC XM_022421319.1 GTCTTCCTTCTCTCTTGGCCATTTACCTGGATTATCGACAAAGAAAACCTTCATTTTGCC XM_005615931.3 GTCTTCCTTCTCTCTTGGCCATTTACCTGGATTATCGACAAAGAAAACCTTCATTTTGCC XM_005615934.3 GTCTTCCTTCTCTCTTGGCCATTTACCTGGATTATCGACAAAGAAAACCTTCATTTTGCC NM_001314029.1 ------NR_052005.1 GTTTCCTTTCCCTCTTGGCCATATTCTGGTGTCAATGACAAGGAGTACCTTGGCTTTGCC NM_014143.3 GTTTCCTTTCCCTCTTGGCCATATTCTGGTGTCAATGACAAGGAGTACCTTGGCTTTGCC NM_001267706.1 GTTTCCTTTCCCTCTTGGCCATATTCTGGTGTCAATGACAAGGAGTACCTTGGCTTTGCC

AB898678.1 ------XM_005615932.3 ACATAGCAAAGCTGAAGAAACAGAGTCTCTAACAGAACTCATTGTTACCTTTT---GTAC XM_005615933.3 ACATAGCAAAGCTGAAGAAACAGAGTCTCTAACAGAACTCATTGTTACCTTTT---GTAC XM_022421251.1 ACATAGCAAAGCTGAAGAAACAGAGTCTCTAACAGAACTCATTGTTACCTTTT---GTAC XM_022421407.1 ACATAGCAAAGCTGAAGAAACAGAGTCTCTAACAGAACTCATTGTTACCTTTT---GTAC XM_022421286.1 ACATAGCAAAGCTGAAGAAACAGAGTCTCTAACAGAACTCATTGTTACCTTTT---GTAC XM_022421319.1 ACATAGCAAAGCTGAAGAAACAGAGTCTCTAACAGAACTCATTGTTACCTTTT---GTAC XM_005615931.3 ACATAGCAAAGCTGAAGAAACAGAGTCTCTAACAGAACTCATTGTTACCTTTT---GTAC XM_005615934.3 ACATAGCAAAGCTGAAGAAACAGAGTCTCTAACAGAACTCATTGTTACCTTTT---GTAC NM_001314029.1 ------NR_052005.1 ACATGTCAAGGCTGAAGAAACAGTGTCTCCAACAGAGCTCCTTGTGTTATCTGTTTGTAC NM_014143.3 ACATGTCAAGGCTGAAGAAACAGTGTCTCCAACAGAGCTCCTTGTGTTATCTGTTTGTAC NM_001267706.1 ACATGTCAAGGCTGAAGAAACAGTGTCTCCAACAGAGCTCCTTGTGTTATCTGTTTGTAC

AB898678.1 ------XM_005615932.3 ATGTTCGTTTGTACGGTGTCCCTCGTTTGCATGACAACATTCT--TTGTGAAGTACAAGG XM_005615933.3 ATGTTCGTTTGTACGGTGTCCCTCGTTTGCATGACAACATTCT--TTGTGAAGTACAAGG XM_022421251.1 ATGTTCGTTTGTACGGTGTCCCTCGTTTGCATGACAACATTCT--TTGTGAAGTACAAGG XM_022421407.1 ATGTTCGTTTGTACGGTGTCCCTCGTTTGCATGACAACATTCT--TTGTGAAGTACAAGG XM_022421286.1 ATGTTCGTTTGTACGGTGTCCCTCGTTTGCATGACAACATTCT--TTGTGAAGTACAAGG XM_022421319.1 ATGTTCGTTTGTACGGTGTCCCTCGTTTGCATGACAACATTCT--TTGTGAAGTACAAGG XM_005615931.3 ATGTTCGTTTGTACGGTGTCCCTCGTTTGCATGACAACATTCT--TTGTGAAGTACAAGG XM_005615934.3 ATGTTCGTTTGTACGGTGTCCCTCGTTTGCATGACAACATTCT--TTGTGAAGTACAAGG NM_001314029.1 ------NR_052005.1 ATGTGCATTTGTACAGTAATTGGT------GTGACAGTGTTCTTTGTGTGAATTACAGGC NM_014143.3 ATGTGCATTTGTACAGTAATTGGT------GTGACAGTGTTCTTTGTGTGAATTACAGGC NM_001267706.1 ATGTGCATTTGTACAGTAATTGGT------GTGACAGTGTTCTTTGTGTGAATTACAGGC

AB898678.1 ------XM_005615932.3 AAGGATTCTGACTG------AGCTTACTCCATTCCT XM_005615933.3 AAGGATTCTGACTG------AGCTTACTCCATTCCT XM_022421251.1 AAGGATTCTGACTG------AGCTTACTCCATTCCT XM_022421407.1 AAGGATTCTGACTG------AGCTTACTCCATTCCT XM_022421286.1 AAGGATTCTGACTG------AGCTTACTCCATTCCT XM_022421319.1 AAGGATTCTGACTG------AGCTTACTCCATTCCT XM_005615931.3 AAGGATTCTGACTG------AGCTTACTCCATTCCT XM_005615934.3 AAGGATTCTGACTG------AGCTTACTCCATTCCT NM_001314029.1 ------NR_052005.1 AAGAATTGTGGCTGAGCAAGGCACATAGTCTACTCAGTCTATTCCTAAGTCCTAACTCCT NM_014143.3 AAGAATTGTGGCTGAGCAAGGCACATAGTCTACTCAGTCTATTCCTAAGTCCTAACTCCT NM_001267706.1 AAGAATTGTGGCTGAGCAAGGCACATAGTCTACTCAGTCTATTCCTAAGTCCTAACTCCT

110

Appendices

AB898678.1 ------XM_005615932.3 CTTTGTGGTGTTGAATTTGTCAGGCACTTTATCC-TCTTGTCTTGTGTTTTACTCTAAAT XM_005615933.3 CTTTGTGGTGTTGAATTTGTCAGGCACTTTATCC-TCTTGTCTTGTGTTTTACTCTAAAT XM_022421251.1 CTTTGTGGTGTTGAATTTGTCAGGCACTTTATCC-TCTTGTCTTGTGTTTTACTCTAAAT XM_022421407.1 CTTTGTGGTGTTGAATTTGTCAGGCACTTTATCC-TCTTGTCTTGTGTTTTACTCTAAAT XM_022421286.1 CTTTGTGGTGTTGAATTTGTCAGGCACTTTATCC-TCTTGTCTTGTGTTTTACTCTAAAT XM_022421319.1 CTTTGTGGTGTTGAATTTGTCAGGCACTTTATCC-TCTTGTCTTGTGTTTTACTCTAAAT XM_005615931.3 CTTTGTGGTGTTGAATTTGTCAGGCACTTTATCC-TCTTGTCTTGTGTTTTACTCTAAAT XM_005615934.3 CTTTGTGGTGTTGAATTTGTCAGGCACTTTATCC-TCTTGTCTTGTGTTTTACTCTAAAT NM_001314029.1 ------NR_052005.1 CCTTGTGGTGTTGGATTTGTAAGGCACTTTATCCCTTTTGTCTCATGTTTCATCGTAAAT NM_014143.3 CCTTGTGGTGTTGGATTTGTAAGGCACTTTATCCCTTTTGTCTCATGTTTCATCGTAAAT NM_001267706.1 CCTTGTGGTGTTGGATTTGTAAGGCACTTTATCCCTTTTGTCTCATGTTTCATCGTAAAT

AB898678.1 ------XM_005615932.3 GACATAGGCAGAAATGATACTTAGTTCTACATTTTAATTGTCATTGTTTACATCTGCATT XM_005615933.3 GACATAGGCAGAAATGATACTTAGTTCTACATTTTAATTGTCATTGTTTACATCTGCATT XM_022421251.1 GACATAGGCAGAAATGATACTTAGTTCTACATTTTAATTGTCATTGTTTACATCTGCATT XM_022421407.1 GACATAGGCAGAAATGATACTTAGTTCTACATTTTAATTGTCATTGTTTACATCTGCATT XM_022421286.1 GACATAGGCAGAAATGATACTTAGTTCTACATTTTAATTGTCATTGTTTACATCTGCATT XM_022421319.1 GACATAGGCAGAAATGATACTTAGTTCTACATTTTAATTGTCATTGTTTACATCTGCATT XM_005615931.3 GACATAGGCAGAAATGATACTTAGTTCTACATTTTAATTGTCATTGTTTACATCTGCATT XM_005615934.3 GACATAGGCAGAAATGATACTTAGTTCTACATTTTAATTGTCATTGTTTACATCTGCATT NM_001314029.1 ------NR_052005.1 GGCATAGGCAGAGATGATACCTAATTCTGCATTTGA-TTGTCACTTTTTGTACCTGCATT NM_014143.3 GGCATAGGCAGAGATGATACCTAATTCTGCATTTGA-TTGTCACTTTTTGTACCTGCATT NM_001267706.1 GGCATAGGCAGAGATGATACCTAATTCTGCATTTGA-TTGTCACTTTTTGTACCTGCATT

AB898678.1 ------XM_005615932.3 AATTTAATAAAATATTTTTATTTATTTTA------XM_005615933.3 AATTTAATAAAATATTTTTATTTATTTTA------XM_022421251.1 AATTTAATAAAATATTTTTATTTATTTTA------XM_022421407.1 AATTTAATAAAATATTTTTATTTATTTTA------XM_022421286.1 AATTTAATAAAATATTTTTATTTATTTTA------XM_022421319.1 AATTTAATAAAATATTTTTATTTATTTTA------XM_005615931.3 AATTTAATAAAATATTTTTATTTATTTTA------XM_005615934.3 AATTTAATAAAATATTTTTATTTATTTTA------NM_001314029.1 ------NR_052005.1 AATTTAATAAAATATTCTTATTTATTTTGTTACTTGGTACACCAGCATGTCCATTTTCTT NM_014143.3 AATTTAATAAAATATTCTTATTTATTTTGTTACTTGGTACACCAGCATGTCCATTTTCTT NM_001267706.1 AATTTAATAAAATATTCTTATTTATTTTGTTACTTGGTACACCAGCATGTCCATTTTCTT

AB898678.1 ------XM_005615932.3 ------XM_005615933.3 ------XM_022421251.1 ------XM_022421407.1 ------XM_022421286.1 ------XM_022421319.1 ------XM_005615931.3 ------XM_005615934.3 ------NM_001314029.1 ------NR_052005.1 GTTTATTTTGTGTTTAATAAAATGTTCAGTTTAACATCCCAGTGGAGAAAGTTAAAAAA NM_014143.3 GTTTATTTTGTGTTTAATAAAATGTTCAGTTTAACATCCCAGTGGAGAAAGTTAAAAAA NM_001267706.1 GTTTATTTTGTGTTTAATAAAATGTTCAGTTTAACATCCCAGTGGAGAAAGTTAAAAAA

111

Appendices

Appendix 6. Sequence alignment of RT-qPCR products with database sequences. Sequence was aligned using Matcher from the EMBOSS suite of programmes.

PD-1

PD1Seg CCCCAACACACAGATCAACGAGAGCCCCCGTGCGGAGCTCACCGTGAAGG :::::::::::::::::::::::: ::::: :: :::::: :::::: :: AB8986 CCCCAACACACAGATCAACGAGAGTCCCCGCGCAGAGCTCTCCGTGACGG 450 460 470 480 490

60 70 80 90 100 PD1Seg AGAGAATCCTGGAGCCCCCCACGGAGAGCCCCAGTCCCCCACCCAGACTC :::::: ::::::::::::::: :::::::::: ::::::::::::::: AB8986 AGAGAACCCTGGAGCCCCCCACACAGAGCCCCAGCCCCCCACCCAGACTC 500 510 520 530 540

110 120 130 140 150 PD1Seg CCTGGCCAGTTACAGGGGTTGGTCATTGGCATCACAGGTGTGCTGGTGGG :::::::: :::::: ::::::: ::: :::: : ::::::::::: AB8986 AGCGGCCAGTTGCAGGGGCTGGTCATCGGCGTCACGAGCGTGCTGGTGGG 550 560 570 580 590

160 170 180 190 PD1Seg GGTCCTACTCCTGCTGCTGCTGACCTGGGTCCTGGCCGCTGTCTTC ::::: :: :::::::::::::::::::::::::::::::::::: AB8986 TGTCCTGCTACTGCTGCTGCTGACCTGGGTCCTGGCCGCTGTCTTC 600 610 620 630

112

Appendices

PD-L1

PDL1se TCTTGGTGTAGTCCTGGCAGTCACTTTTTGTTTAAAAAAACATGGGAGAA ::::::::::::::::::::::::::: ::: :::::::::::::::::: AB8986 TCTTGGTGTAGTCCTGGCAGTCACTTTCTGTCTAAAAAAACATGGGAGAA 820 830 840 850 860

60 70 80 90 100 PDL1se TGATGGATGTGGAAAAATGTTGCACCCGAGATAGGAACTCAAAGAAACGA :::::::::::::::::::::::::::::::::::::::::::::::::: AB8986 TGATGGATGTGGAAAAATGTTGCACCCGAGATAGGAACTCAAAGAAACGA 870 880 890 900 910

110 120 130 140 150 PDL1se AATGATATACAATTTGAAGAGACATAATCCAGCATGGAAACTCCTGATCT :::::::::::::::::::::::::::::::::::::::::::::::::: AB8986 AATGATATACAATTTGAAGAGACATAATCCAGCATGGAAACTCCTGATCT 920 930 940 950 960

160 170 180 PDL1se TAAAGCAGGGATTCNCGGCCTGTGNTTTGANTTCAG :::::::::::::: ::::::::: ::::: ::::: AB8986 TAAAGCAGGGATTCTCGGCCTGTGGTTTGAGTTCAG 970 980 990 1000

GUSB

GUSBse AGACGCTTCCA-GTACCCCAAGGGTTACTTCGTCCAGAACACATACTTTG ::::::::::: :::::::::::::::::::::::::::::::::::::: NM_001 AGACGCTTCCAAGTACCCCAAGGGTTACTTCGTCCAGAACACATACTTTG 630 640 650 660 670

50 60 70 80 90 GUSBse ACTTCTTCAACTACGCGGGCCTGCATCGCCCTGTGCTCCTCTACACCACA :::::::::::::::::::::::::::::::::::::::::::::::::: NM_001 ACTTCTTCAACTACGCGGGCCTGCATCGCCCTGTGCTCCTCTACACCACA 680 690 700 710 720

100 GUSBse CC :: NM_001 CC 730

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Appendices

HPRT-1 from spleen

HPRT1_ TTGCTGGTGAAAAGGACCCCCTGATGTTTTAGTTATAAATCAGACTTTGT :::::::::::::::::::: :: :: :: : :::::: : :::::::: NM_001 TTGCTGGTGAAAAGGACCCCTCGAAGTGTTGGCTATAAACCTGACTTTGT 640 650 660 670 680

60 70 80 90 100 HPRT1_ TAGGATTTGAA--TCCAGACAAGTTTATTGTAGGATATGCCCTTGACTAT : ::::::::: ::::::::::::: ::::::::::::::::::::::: NM_001 T-GGATTTGAAATTCCAGACAAGTTTGTTGTAGGATATGCCCTTGACTAT 690 700 710 720 730

HPRT1_ AA :: NM_001 AA

RPS5

RPS5se TCACTGGTGAGA-CCCCCTGCAGGTCCTGGTGAATGCCATTATTAACAGT :::::::::::: ::::::::::::::::::::::::::::::::::::: XM_533 TCACTGGTGAGAACCCCCTGCAGGTCCTGGTGAATGCCATTATTAACAGT 450 460 470 480 490

50 60 70 80 90 RPS5se GGTCCCCGGGAAGACTCAACCCGCATTGGGCGAGCTGGAACAGTGAGGCG :::::::::::::::::::::::::::::::::::::::::::::::::: XM_533 GGTCCCCGGGAAGACTCAACCCGCATTGGGCGAGCTGGAACAGTGAGGCG 500 510 520 530 540

100 110 120 130 140 RPS5se TCAGGCTGTCGATGTGTCCCCCCTACGCCGTGTGAATCAGG ::::::::::::::::::::::::::::::::::::::::: XM_533 TCAGGCTGTCGATGTGTCCCCCCTACGCCGTGTGAATCAGG 550 560 570 580

114

Appendices

RPS19

RPS19s CCTTCCTCAAAA-GTCTGGGAAGCTGAAAGTCCCTGAATGGGTGGACACT :::::::::::: ::::::::::::::::::::::::::::::::::::: XM_005 CCTTCCTCAAAAAGTCTGGGAAGCTGAAAGTCCCTGAATGGGTGGACACT 210 220 230 240 250

50 60 70 80 90 RPS19s GTCA-GCTGGCCAAGCATAAAGAGCTTGCTCCCTACGATGAGAAC :::: :::::::::::::::::::::::::::::::::::::::: XM_005 GTCAAGCTGGCCAAGCATAAAGAGCTTGCTCCCTACGATGAGAAC 260 270 280 290

115