modulating the cd8 papillomavirus-induced lesions: … · 2017-12-21 · arranja sempre que eu...

Dissertação de candidatura ao grau de Mestre em Oncologia – especialização em Oncologia Molecular – submetida ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Porto. Orientador – Professor Doutor Rui Gil da Costa Categoria – Investigador Afiliação – LEPABE, Faculdade de Engenharia da Universidade do Porto; Grupo de Oncologia Molecular e Patologia Viral, CI-IPOP, Instituto Português de Oncologia do Porto Co-Orientador – Professor Doutor Rui Medeiros Categoria – Professor Convidado Associado Afiliação – Instituto de Ciências Biomédicas de Abel Salazar; Grupo de Oncologia Molecular e Patologia Viral, CI-IPOP, Instituto Português de Oncologia do Porto Co-Orientador – Professor Doutor Manuel Vilanova Categoria – Professor Associado Afiliação – Instituto de Ciências Biomédicas de Abel Salazar

CARLOS EDUARDO REIS DOS SANTOS

MODULATING THE CD8+ T CELL RESPONSE IN HUMAN PAPILLOMAVIRUS-INDUCED LESIONS: STUDIES IN THE K14-HPV16 TRANSGENIC MOUSE MODEL

III

Alguns dos resultados incluídos nesta dissertação de mestrado foram submetidos

para publicação:

- Santos C., Ferreirinha P., Sousa H., Ribeiro J., Bastos M.M.S.M., Faustino-Rocha

A.I., Oliveira P.A., Medeiros R., Vilanova M., Gil da Costa R.M. (2015) Kinetics of CD8+ T

cells in human papillomavirus-induced lesions – data from K14-HPV16 transgenic mice.

Submitted to the journal Tumor Biology with the number TUBI-D-15-04094.

- Santos C., Neto T., Ferrerinha P., Sousa H., Ribeiro J., Bastos M.M.S.M., Oliveira

P.A., Medeiros R., Vilanova M., Gil da Costa R.M. (2015) Celecoxib promotes

degranulation of CD8+ T cells in HPV-induced lesions of transgenic mice. Submitted to the

journal Antiviral Research with the number AVR-D-16-00002.

Alguns resultados foram também publicados em atas de um congresso internacional:

- Santos C., Ferreirinha P., Sousa H., Ribeiro J., Bastos M.M.S.M., Faustino-Rocha

A.I., Oliveira P.A., Medeiros R., Vilanova M., Gil da Costa R.M. (2015) Role of CD8+ T

cells in HPV-induced skin cancer: data from a K14-HPV16 transgenic mouse model;

Proceedings of the HPV2015 – 30th International Papillomavirus Conference & Clinical

and Public Health Workshops; 17th-21st September 2015, Lisbon, Portugal. (Best poster

presentation award)

V

“When you want to succeed as bad as you want to bre athe then you’ll be successful” – Eric Thomas

VII

Agradecimentos Neste momento, que marca o final de mais uma etapa da minha vida, não poderia

deixar de agredecer às pessoas que de alguma forma contribuíram para a conclusão

desta etapa. Este último ano foi bastante desafiante e exigente e todo o conhecimento e

apoio que recebi foi fundamental para chegar até aqui.

Em primeiro lugar, gostaria de agradecer à pessoa que me recebeu e que me

acompanhou mais de perto no desenvolvimento deste trabalho, o meu orientador,

Professor Doutor Rui Gil da Costa. Agradeço a sua confiança em mim para levar a cabo

este trabalho, todos os conhecimentos que me transmitiu, a disponibilidade para ajudar

no que fosse preciso, todos os conselhos e a constante boa disposição.

Ao Professor Doutor Rui Medeiros, meu co-orientador, agradeço toda a ajuda

fornecida ao longo deste ano e por tudo o que me ensinou. Mesmo estando cheio de

trabalho arranjava maneira de me receber sempre que aparecia no seu gabinete do IPO

para me poder ajudar no que eu precisasse e sempre bem-disposto.

Ao Professor Doutor Manuel Vilanova, também meu co-orientador, por tudo aquilo

que continua a ensinar-me, pelo seu rigor que tanto aprecio, pela disponibilidade que

arranja sempre que eu preciso de falar consigo mesmo quando a quantidade de trabalho

não o “deixa respirar”.

A este conjunto de orientadores agradeço todas as discussões das quais surgiram

ideias de trabalho, agradeço tudo o que me ensinaram e, acima de tudo, agradeço a

compreensão e o apoio que sempre demonstraram perante a minha vida de estudante-

atleta. Não só compreendem como também valorizam o que faço e é realmente bom

sentir esse apoio.

Ao Pedro Ferreirinha, mais do que um co-orientador no passado, um amigo e

colega investigador no presente. Muito do que aprendi sobre trabalhar num laboratório

devo-o a ele. Para além de tudo o que me ensinou e continua a ensinar está sempre

disposto a ajudar no que for preciso. Não só é dedicado ao trabalho como também é uma

pessoa óptima para ir tomar um café e ter uma conversa animada.

Às restantes pessoas que trabalham no Laboratório de Imunologia Mário Arala

Chaves (ICBAS), em especial à Encarnação Rebelo, Joana Alves e Alexandra Correia,

agradeço o carinho e a boa disposição de todos os dias, a disponibilidade para ajudar no

que precisasse e as conversas alegres e enriquecedoras.

À Professora Paula Oliveira, à Ana Faustino-Rocha e ao Tiago Neto (todos da

Universidade de Trás-os-Montes e Alto Douro) agradeço todo o apoio que me deram

durante a realização dos trabalhos experimentais.

VIII

Por último, mas muito importantes, a família e os amigos formam um conjunto de

pilares que servem de apoio nos momentos mais complicados e de diversão quando a

ocasião o permite. Àqueles que me acompanharam mais de perto durante este ano

mesmo, em alguns casos, estando longe (Tiago Silva, Beatriz Silva, Helena Pinheiro,

Mariana Pereira, Inês Sousa, Hildeberto Moreira, Ana Rosa, Luciana Leite, Rita Faria,

Miguel Gonçalves, Cátia Oliveira, Joana Pinto, Renata Lopes, Diogo Edi, Isabel Paiva,

Catarina Pereira e, claro, aos meus colegas de equipa) agradeço pela força que me

deram quando as coisas pareciam não correr bem, a motivação que me passaram

quando esta me faltava, as conversas enriquecedoras sobre o trabalho, os momentos de

descontração, gargalhada e diversão quando estes eram possíveis e, principalmente, por

serem verdadeiros amigos e compreenderem os sacrifícios que faço em prol dos meus

objectivos e ambições enquanto estudante-atleta.

Aos meus pais e irmã, agradeço por tudo aquilo que não é possível agradecer o

suficiente. Agradeço pela educação que me deram e por tudo o que me ensinaram, por

todas as condições que sempre me deram para o meu crescimento, pelas chamadas de

atenção, por exigirem o melhor de mim, por me apoiarem em todas as decisões, por me

orientarem. Se hoje posso dizer que tenho orgulho no que me tornei devo-o inteiramente

a eles. Obrigado.

IX

Index

Agradecimentos .............................................................................................................. VII

Index ................................................................................................................................ IX

Abbreviations ................................................................................................................... XI

Resumo ......................................................................................................................... XIII

Abstract .......................................................................................................................... XV

Chapter 1: Introduction ..................................................................................................... 1

The Basis of HPV Infection and Cell Transformation ..................................................... 4

HPV Infection vs. Host Immune Response .................................................................... 5

HPV-associated Chronic Inflammation .......................................................................... 6

HPV-induced Carcinogenesis: The Best Models to Understand It ................................. 9

K14-HPV16 Transgenic Mice as a Model for Inflammation-associated Carcinogenesis12

Modulators of the immune response and their effect over CD8+ T cells ........................15

Chapter 2: Objectives ......................................................................................................17

Chapter 3: Kinetics of CD8+ T cells in human papillomavirus-induced lesions..................19

Introduction ..................................................................................................................21

Material & Methods ......................................................................................................22

Animals .....................................................................................................................22

Mice genotyping........................................................................................................22

Study Design ............................................................................................................22

Histology ...................................................................................................................22

Preparation of Single-Cell Suspensions ....................................................................23

Immunophenotyping .................................................................................................23

Statistical Analysis ....................................................................................................23

Results .........................................................................................................................24

Transgenic Mice Show Epidermal Hyperplasia and Dysplasia ..................................24

Increased CD8+ T lymphocytes numbers and activation in HPV16+/- mice ................24

Discussion ....................................................................................................................26

Conclusion ...................................................................................................................27

Chapter 4: Ptaquiloside inhibits tumour-infiltrating CD8+ T cells in HPV-transgenic mice .29

Introduction ..................................................................................................................31


Mice ..........................................................................................................................32

X

Mice genotyping........................................................................................................32

Ptaquiloside isolation ................................................................................................32

Study Design ............................................................................................................33

Ptaquiloside administration and toxicity ....................................................................33

Skin histology ...........................................................................................................34

Isolation of a Single-Cell Suspension ........................................................................34

Flow Cytometry Analysis ...........................................................................................34


Results .........................................................................................................................35

General findings........................................................................................................35

Cutaneous lesions ....................................................................................................37

CD8+ T lymphocytes are present in ptaquiloside-treated and untreated animals .......37

Ptaquiloside decreases the number of CD8+CD107a+ T cells in HPV16+/- mice ........38

Reduced number of CD8+CD44+ T cells in ptaquiloside-treated transgenic animals .39

Discussion ....................................................................................................................40

Conclusion ...................................................................................................................42

Chapter 5: Celecoxib promotes degranulation of CD8+ T cells in HPV-induced lesions of transgenic mice ................................................................................................................45

Introduction ..................................................................................................................47


Animals .....................................................................................................................48

Mice genotyping........................................................................................................48

Experimental Design .................................................................................................48

Celecoxib administration ...........................................................................................49

Histological Analysis .................................................................................................49

Preparation of Single-Cell Suspensions ....................................................................49

Immunophenotyping .................................................................................................49


Results .........................................................................................................................50

General findings........................................................................................................50

Histological analysis .................................................................................................50

CTL infiltration and activation in celecoxib-treated mice ............................................51

Discussion ....................................................................................................................53

Conclusion ...................................................................................................................54

Chapter 6: General Discussion and Conclusions .............................................................55

References ......................................................................................................................61

XI

Abbreviations 4-NQO – 4-Nitroquinoline 1-oxide

AP-1 – Activator Protein-1

APC – Antigen Presenting Cell

BPV – Bovine Papillomavirus

CD – Cluster of Differentiation

CIN – Cervial Intraepithelial Neoplasia

COX – Cyclooxygenase

CpG – cytosine-phosphate-guanine motif

CPV – Canine Papillomavirus

CRPV – Cottontail Rabbit Papillomavirus

CTL – Cytotoxic T Lymphocyte

CXB – Celecoxib

CXCL12 – C-X-C motif chemokine 12

DC – Dendritic Cell

FASL – FAS Ligand

FcγR – Fragment crystallizable gamma Receptor

H&E – Haematoxilin-Eosin

HIF-1 – Hypoxia Inducible Factor-1

HNSCC – Head and Neck Squamous Cell Carcinoma

HPV – Human Papillomavirus

IFN – Interferon

Ig – Immunoglobulin

IL – Interleukin

K14 – Keratin-14

LAMP1 – Lysosome-associated membrane protein 1

mAb – Monoclonal Antibody

XII

MC – Mast Cell

MHC – Major Histocompatibility Complex

miR – MicroRNA

MMP – Matrix Metalloproteinase

N – North

NF-κB – Nuclear Factor kappa-B

NK – Natural Killer

NMR – Nuclear Magnetic Resonance

ORF – Open Reading Frame

pRb – Retinoblastoma protein

PRR – Pattern Recognition Receptor

PTQ – Ptaquiloside

SCC – Squamous Cell Carcinoma

TAM – Tumour-associated Macrophage

TAM67 - Dominant-negative c-Jun

Th – T helper

TIMP – Tissue Inhibitor of Metalloproteinase

TLR – Toll-like Receptor

TNF-α – Tumour Necrosis Factor-alpha

TRAIL – TNF-related Apoptosis-Inducing Ligand

Treg – T regulatory

VEGF – Vascular Endothelial Growth Factor

VLP – Virus Like Particle

W – West

WT – Wild-type

XIII

Resumo O Papilomavirus Humano (HPV) consiste em pequenos fragmentos circulares de

ADN de dupla cadeia sem invólucro, associado ao desenvolvimento de lesões benignas e

malignas na pele e mucosas queratinizadas. O murganho transgénico K14-HPV16 é um

modelo útil para estudar o processo de carcinogénese induzido por HPV, dado que se

assemelha ao processo que acontece em doentes oncológicos. Este processo é

acompanhado por um complexo infiltrado de leucócitos que desempenham um papel

decisivo quer na progressão quer na regressão das lesões. Os linfócitos T CD8+

citotóxicos (CTL) são uma parte importante deste infiltrado dado que são capazes de

destruir células infectadas por vírus e células tumorais. Neste trabalho, a infiltração e

actividade dos CTL foi analisada em diferentes tempos de vida e diferentes lesões. Ainda,

foi analisado o efeito de dois imuno-moduladores – uma toxina imunosupressora

(ptaquilosídeo – PTQ) e um fármaco anti-inflamatório (celecoxib – CXB, um inibidor

selectivo da ciclooxigenase-2) – na infiltração das células T CD8+ e na sua actividade.

Após o sacrifício dos murganhos, foram colhidas amostras de pele do peito de

murganhos wild-type (WT, HPV-/-) e transgénicos (HPV+/-), que foram tratados com PTQ,

com CXB ou não receberam tratamento. Os fragmentos de pele foram digeridos com

colagenase e a suspensão celular obtida foi analisada por citometria de fluxo. Para além

disso, foram colhidas amostras de pele correspondentes para análise histológica.

Todos os murganhos HPV-/- apresentam histologia da pele normal enquanto todos

os animais HPV+/- exibem lesões cuja agressividade aumenta através de etapas etárias

consecutivas. Verificou-se que os murganhos transgénicos apresentam maior infiltração e

actividade de células T CD8+ quando comparados com animais WT da mesma idade e,

que a activação destas células aumenta com a idade e acompanha a progressão das

lesões. Foram também estudados os efeitos de uma toxina de origem vegetal, o

ptaquilosídeo, que se pensa contribuir para a carcinogénese induzida por papilomavírus

em animais e em populações humanas. Os murganhos transgénicos tratados com PTQ

mostram menor percentagem de linfócitos T CD8+ activados quando comparados com os

animais HPV+/- não tratados, revelando um novo efeito imunossupressor desta toxina. Por

outro lado, os murganhos transgénicos tratados com CXB apresentam menor infiltração

de células citotóxicas comparativamente com os animais não tratados, mas apresentam

um aumento da percentagem de linfócitos T CD8+ activados. Esta observação sugere

possíveis abordagens terapêuticas às neoplasias induzidas pelo HPV, nomeadamente o

uso de inibidores selectivos da ciclooxigenase-2 para potenciar a actividade dos CTL.

XIV

Globalmente, este trabalho levanta diversas questões sobre a função das células

T CD8+ nos cancros induzidos por HPV, abrindo o caminho para futuros estudos na área

da imunotoxicologia e da imunoterapia.

XV

Abstract Human papillomavirus (HPV) is a small non-enveloped double-stranded circular

DNA virus associated with the development of cutaneous and mucosal malignancies. The

K14-HPV16 transgenic mouse is an useful model to study the process of carcinogenesis

induced by a HPV infection as it closely mimics the occurring process in human patients.

This process is accompanied by a complex immune cell infiltrate which plays a key role in

either lesion progression or regression. Cytotoxic CD8+ T lymphocytes (CTL) are an

important part of this infiltrate as they target virus-infected and tumour cells for

destruction. Thus, in this work, CTL infiltration and activity was assessed and compared at

different time-points. In addition, the effect of two distinct immunomodulators – an

immunosuppressive toxin (ptaquiloside – PTQ) and an anti-inflammatory drug (celecoxib –

CXB, a selective inhibitor of cyclooxygenase-2) – over CD8+ T cell infiltration and activity

was examined.

Following the mice sacrifice, chest skin samples were collected from untreated,

PTQ-treated or CXB-treated wild-type (WT, HPV-/-) and transgenic (HPV+/-) mice. Skin

fragments were digested with collagenase and the obtained cell suspension was analysed

by flow cytometry. Additionally, matched skin samples were histologically examined.

All HPV-/- mice show normal skin histology whilst all HPV+/- animals present skin

lesions, with increased aggressiveness in older and PTQ-treated mice. Untreated

transgenic mice show increased CTL infiltration and activity when compared to untreated

WT animals. CD8+ T cell activation is higher in older HPV+/- mice than in younger

transgenic mice. Moreover, transgenic mice receiving PTQ treatment show reduced

percentage of activated CTL when comparing with untreated HPV+/- animals. Furthermore,

CXB-treated mice presented lower infiltration of cytotoxic cells when compared with the

respective controls. Despite the reduction regarding infiltrating cells, CXB-treated HPV+/-

mice showed increased percentage of activated CD8+ T lymphocytes than controls.

These findings suggest that CTL infiltrate HPV-induced lesions and their activity is

enhanced in more aggressive lesions. Moreover, the trafficking and activity of these cells

can be modified when exposed to immunomodulators. In this regard, ptaquiloside seems

to decrease CTL activity while celecoxib appears to enhance this activity.

1

Chapter 1: Introduction

3

Human papillomavirus (HPV) is the pathogen responsible for the most common

sexually transmitted infection among both men and women in the United States [1]. Most

sexually active people will be infected by HPV during their life. The vast majority of HPV

infections in immunocompetent individuals are cleared by the immune system, but in

approximately 10% of cases [2, 3] the persistence of this infection will cause the

development of disease and progression to cancer. HPV has tropism for skin and mucosal

epithelia especially from the anogenital and oropharyngeal areas. High-risk HPVs (mostly

types 16, 18, 31 and 33) act as crucial etiologic agents in cervical carcinoma [4] – which is

the sixth most frequent malignancy in women worldwide [5] – and are the cause of some

head and neck neoplasms where incidence depends on the country and other associated

risk factors like tobacco [6, 7].

Since 1863 [8], when Virchow first hypothesised a link between inflammation and

cancer, many studies have been carried out in order to understand the underlying

mechanisms of this relation and its intervenients. The importance of this relation was

further reinforced by the award of the Nobel Prize to Robin Warren and Barry Marshall for

their work in discovering Helicobacter pylori as the etiologic agent of inflammation-

associated gastric cancer [9]. The tumour microenvironment is characterised by the

presence of a variety of leucocytes which have been shown to be attracted by

chemokines produced by tumour and stromal cells [10]. Lymphocytes are an important

part of inflammatory infiltrates in the tumour microenvironment [8, 11] and different

lymphocytic populations can play different roles. For instance, T helper (Th) 1, Tγδ and

cytotoxic T cells are associated with disease-free survival. In contrary, Th2, Th17 and T

regulatory (Treg) cells are allied with a poorer outcome [12]. Neutrophils, tumour-

associated macrophages (TAMs), dendritic cells (DCs) and mast cells represent other

leucocyte populations also frequently found in the tumour environment that may play dual

roles during carcinogenesis [8, 13]. These tumour-associated inflammatory cells, together

with the tumour cells, produce and secrete a wide set of cytokines (e.g.: tumour necrosis

factor (TNF)-α, interleukin (IL)-1, IL-6 and monocyte colony-stimulating factor) and

chemokines (e.g.: monocyte chemotactic protein-1) that may either help tumour

progression or contain it [14-17].

In the case of HPV-associated carcinogenesis, the virus infects keratinocytes and

an immunological response arises as cytokines (mostly TNF-α, IL-1 and type I and II

interferons – IFNs) are released by the infected keratinocytes and local immune cells [18].

However, HPVs may avoid the effector functions of pro-inflammatory cytokines by

different mechanisms [19-22]. Thus, if the infection persists, the chance of neoplastic

transformation rises [23]. Besides its importance in the early stages of carcinogenesis,

4

innate and adaptive immune responses are critical factors involved in malignant

progression.

The Basis of HPV Infection and Cell Transformation

Human papillomaviruses have their genome divided in three main regions: the

non-coding upper regulatory region, the early region encoding six known oncoproteins

and the late region, which encodes the capsid proteins. The oncoproteins encoded in the

early region, mainly E6 and E7 but also E5, are key participants in the infectious cycle and

are the source of the genetic alterations which potentially lead to HPV-associated

carcinogenesis as reviewed elsewhere [24]. The E5 oncogene is believed to have low

impact in the maintenance of the malignant phenotype of cervical cancer cells as it

appears to be deleted in various carcinomas [25]. Nevertheless, when present, it takes an

important part in multiple mechanisms favouring neoplastic transformation. Some in vitro

studies have shown that the HPV16 E5 oncoprotein can promote cell cycle progression

and DNA synthesis by down-regulating the expression of tumour suppressor proteins p21

and p27 [26, 27]. Moreover, this oncoprotein is central to evasion of the host

immunosurveillance. In E5-expressing cell lines antigen-presenting major

histocompatibility complex (MHC) class I molecules appear to be trapped in the Golgi

apparatus which reduced their surface expression [28, 29]. The down-regulation of MHC

class I on cells expressing E5 ultimately impairs their recognition and clearance by

cytotoxic T lymphocytes (CTL). The early genes E6 and E7 are known to have great

oncogenic potential. They play a pivotal role in HPV-mediated carcinogenesis as they

react with important tumour suppressor proteins impairing their functions. Thus, E6 binds

to and promotes the degradation of the p53 tumour suppressor protein [30, 31], impairing

important mechanisms of defence such as DNA repair and apoptosis. Additionally, the E6

protein facilitates carcinogenesis by reducing cellular senescence (as a result of

telomerase activation [32]), activating the NF-κB pathway and evading the interferon

response [24]. The E7 oncoprotein binds to tumour suppressor proteins of the

retinoblastoma family of proteins (pRb) and induces their inactivation [33, 34], therefore

increasing DNA synthesis and boosting cellular proliferation. Furthermore, E7 is able to

promote genomic instability, which increases the likelihood of malignant progression [24].

Together, the E6 and E7 oncogenes are capable of transforming and induce the

immortalization of keratinocytes [35]. Until today, the three early proteins mentioned

above appear to be, of all HPV proteins, the ones with higher oncogenic potential. Given

their roles in evading host immunity, it is important to understand how the human immune

system responds to HPV infection.

5

HPV Infection vs. Host Immune Response

Along the years, mammals have grown complex innate and adaptive immune

mechanisms to control autoimmunity and to protect the host against microbial infections.

Viruses can be detected by cellular receptors generally designated as pattern recognition

receptors (PRRs), which trigger innate immune mechanisms that subsequently may be

followed by a more specific adaptive immune response. These cellular receptors are

proficient at recognising several pathogen-associated molecular patterns, like viral DNA or

RNA or bacterial cell wall or intracellular components, and also interacting with danger-

associated molecular patterns like intracellular molecules released from damaged cells

and cells undergoing unprogrammed cell death [36]. HPVs are double-stranded DNA

viruses that are most likely to be recognized by Toll-like receptor 9 (TLR-9). This receptor

is intracellularly expressed in the membrane of endolysosomes and recognizes

unmethylated CpG-DNA sequences harboured by pathogens like viruses, bacteria and

protozoa [36]. However, HPVs appear to somehow surpass these PRRs. The

oncoproteins E6 and E7 may promote the down-regulation of TLR-9 as has been shown

in vitro for HPV16 [37]. Therefore, at the start, the HPV infection seems to occur without

the awareness of the host immune system [38]. Furthermore, in case of a persistent

infection, the virus impairs immune cell functions as well as several molecular pathways

involved in the immune response [39]. Also, other innate immune mechanisms, like the

IFN response, can be abrogated by the actions of some HPV oncoproteins, as mentioned

above.

HPV is transmitted through skin-to-skin contact, usually through micro wounds.

There exists a variety of immune cells divided between the dermis and epidermis. In the

epidermis, the main cellular population are keratinocytes but there can also be found

Langerhans cells and T lymphocytes [39]. Keratinocytes, the main targets of HPV, are

capable of some immune functions. These cells can produce and release cytokines,

induce the activation of memory T cells and may also act as non-professional antigen-

presenting cells (APCs) [40]. Hence, keratinocytes are of great importance in the initiation

of an immune response against HPV infections. On the other hand, the majority of the so-

called “classical” immune cells (macrophages, DCs, T lymphocytes and others) can be

found in the dermis [39]. Both keratinocytes and immune cells on site are capable of pro-

inflammatory cytokine production [41] and antigen presentation in response to several

pathogens. Although the innate immune response to HPV infections remains poorly

understood, the immunological processes described above are thought to be the main

ones responsible for the initiation of an adaptive immune response, which will determine

the clearance or persistence of the infection. In fact, only about 10% of HPV infections will

6

develop clinical symptoms and possibly evolve to cancer [2, 3]. Therefore, warts and low

grade cervical intraepithelial neoplasms (CIN1) are in most cases forced to regress by the

cell-mediated adaptive immune response [42]. It has been shown that CD4+ and CD8+

HPV16-specific T cells migrate to the skin after intradermal challenge with HPV16

peptides [43]. This cellular migration event appears to be followed by an antibody-

mediated response, as observed in HPV-infected women [44]. Still, antibody titres are low

and many women may even not produce them.

HPV-associated Chronic Inflammation

The above described mechanisms are thought to explain how a human immune

system reacts since the beginning of a HPV infection until the moment of immunological

clearance. However, as seen earlier, HPV oncoproteins have ways of evading the host

immune response. This evasion favours the persistence of the infection, which in turn may

promote tumourigenesis.

In this context, the cellular content of the inflammatory infiltrate is decisive for

either neoplastic transformation or elimination of infected/transformed cells (Fig. 1). While

some cellular populations support HPV’s immune evasion and cell transformation

mechanisms, others have a role in inhibiting malignant progression or even in potentiating

tumour regression. For instance, T regulatory (Treg) cells facilitate the viral immune

evasion. These cells display the CD4+CD25+FoxP3+ phenotype and play an

immunosuppressive role important to prevent self-aggression by the immune system [45].

Because of their immunosuppressive function, Treg cells are often associated to

persistent infection and tumour progression. Indeed, studies have shown a correlation

between increased frequencies of FoxP3+ T cells at both systemic [46] and cervical [47]

level and HPV persistent infection. Furthermore, a study has shown the chemokine

CXCL12 (which is not expressed in normal skin) to act as a chemoattractant for Treg cells

[48]. This work shows evidence that HPV stimulates an increased production of CXCL12

which draws a rising number of FoxP3+ cells. Besides Treg cells, another subset of CD4+

T cells can have pro-tumour functions. Type-2 helper T (Th2) cells can also contribute to

impair the protective immune response, predominantly of the Th1 type, in the tumour

microenvironment as they produce IL-4, -5 and -13, as well as IL-10. As seen earlier, this

type of immune response facilitates malignant progression [12]. Furthermore,

macrophages are thought to be a major component of tumour infiltrates [8] and are highly

frequent in HPV-associated tumours [49]. In the presence of Th2-type cytokines like IL-4

and IL-13, tumour-associated macrophages (TAMs) are alternatively activated and

acquire the M2-phenotype [50]. Unlike the M1 macrophages, these M2 cells express

7

various features and functions that are commonly associated with immune regulation and

tumour progression. For instance, M2 TAMs, together with mast cells, are an important

source of growth factors (e.g.: vascular endothelial growth factor – VEGF), cytokines (e.g.:

TNF-α, transforming growth factor-β), proteases (e.g.: matrix metalloproteinases) and

other molecules which in turn promote angiogenesis and tissue remodelling [51, 52].

These events facilitate the intake of nutrients and provide other stimuli which help tumour

growth, invasion and metastasis. On the other hand, TAMs can be classically activated

upon microbial stimuli or induced by pro-inflammatory cytokines, such as IFN-γ, to play

important anti-tumour functions [50]. M1-type TAMs can, therefore, act as inducer and

effector cells in Th1-type immune responses against tumours. They usually display an IL-

12high, IL-23high phenotype and produce high levels of pro-inflammatory cytokines (IL-1β,

IL-6 and TNF-α) and reactive nitrogen species [53]. Besides M1 TAMs, other cellular

populations play important anti-tumour roles among the largely heterogeneous tumour

inflammatory infiltrates. T lymphocytes are also important immune effectors. Type-1

responses, mediated by Th1 cells represent the prototypic response adequate to

eradicate cancer cells. The differentiation of this type of CD4+ T cells is driven by IL-12

and they are characterised by the production of pro-inflammatory cytokines like IFN-γ. Th1

cancer cells can directly induce the killing of tumour cells through TNF-related apoptosis-

inducing ligand (TRAIL) and/or Fas Ligand (FasL) pathways [54] and can induce the

cytotoxic activity of CD8+ T lymphocytes and other effector cells through the production of

specific cytokines [55]. In turn, CD8+ T cells, which may differentiate into cytotoxic T

lymphocytes (CTL), are commonly protective against intracellular pathogens, like viruses,

and tumour cells. These lymphocytes were observed in higher frequency in HPV-positive

carcinomas when compared with HPV-negative lesions [56]. Further, this difference

correlates with the patients’ prognosis as a higher CD8+ T cell frequency is associated

with augmented overall survival. Moreover, “natural killer” (NK) cells and NKT cells act

similarly to CTL but are part of the innate immune response. Unlike CTL, NK cells do not

require the presence of MHC I molecules on target cells to exert their cytotoxic function.

Thus, if HPV successfully down-regulates MHC I expression, thereby avoiding the action

of CTL, infected cells may still be targeted by NK cells. Like CTL, NK and NKT cells

express IFN-γ, perforin and granzyme and are thus capable effectors in the anti-viral

immune response [39] as well as being able to also participate in the killing of cancer cells

[57]. Additionally, several APCs (like DCs and Langerhans cells) are of great value and

are frequent in the tumour microenvironment. They are essential for the activation of

effector cells through antigen presentation and cytokine production [13].

8

Fig. 1: Schematic representation of the immune cell infiltr ate in HPV- induced carcinogenesis. From top to bottom, HPV infection induces neoplastic transformation with associated tissue remodelling events which lea d to host immune response. This response could eit her contribute to disease progression or regression which is reflected in the cellular populations recruited in either case. CD8 +, Th1-type CD4 + T, NK cells and M1 macrophages are commonly associa ted with tumour regression while, Treg, Th2-type CD4 + T cells and M2 macrophages are usually related to malignant progression.

9

The cross-talk of immune cells with HPV-infected cells is still not fully understood.

However, it is generally agreed that the inflammation associated with the viral infection

facilitates the development of HPV-associated cancers. In general terms, regulatory T

cells, M2 TAMs, mast cells and Th2 cells most likely represent the immune cells with the

greatest contribution to persistent infections and malignant progression. Additionally,

HPV-transformed keratinocytes were shown to produce the immunosuppressive cytokine

IL-10 in HPV-associated cervical cancer [58], which may be triggered by the virus

oncoproteins as an immune evasion mechanism. On the other hand, M1 macrophages,

NK and NKT cells, CTL and Th1 cells are, with the support of APCs, the major

participants in anti-viral responses and tumour regression. Still, it is important to

emphasize that this whole interaction in the tumour milieu is greatly complex due to the

heterogeneity of cellular populations therein, which likely justifies the difficulty in

characterising the relationship between inflammatory cells and HPV infection.

HPV-induced Carcinogenesis: The Best Models to Unde rstand It

Along the years, scientists have been improving our understanding of chronic HPV

infection and its association with carcinogenesis. In vitro and in vivo animal models are

key elements towards the evolution of scientific knowledge (Table 1) and the in vitro ones

are already widely used in HPV-induced carcinogenesis research. HPV-related in vitro

studies include, in most cases, the use of single-layer cultures of common cervical

carcinoma cell lines (HeLa, SiHa, CaSki and others). In the last two decades, a more

sophisticated type of in vitro assay was developed and used as an alternative model.

Organotypic (or raft) cultures allow the proliferation and differentiation of epithelial cells at

an air-liquid interface on a dermal-equivalent support [59-61]. Thus, these raft cultures are

useful to study the events occurring in human stratified epithelia in the course of HPV

infection and HPV-induced carcinogenesis.

Alongside with the cell culture technique, in vivo animal models are extensively

exploited for scientific purposes. On one hand, bovine cattle, dogs and rabbits can be

used to study bovine, canine oral and cottontail rabbit papillomaviruses, respectively [62,

63]. The study of these animal papillomaviruses is important as they are etiologic agents

of diseases in farm and companion animals, provide in vivo models for HPV research and

help to discover new subjects to study on HPV. However, these large animals are not

common objects of study because they are associated with a great deal of financial costs

and ethical issues. In turn, the mouse is the most commonly used test subject in health-

associated studies. Mice display a great similarity to humans in terms of anatomy,

physiology, including the immune one, and genetics which, in addition to its cost-

10

effectiveness and a wide set of other advantages, make it applicable for the study of

human diseases [64]. In the past couple of decades, new HPV transgenic mouse models

have been created to more accurately infer on the function of some virus’ oncogenic

proteins. The first transgenic mice were reported in 1993 and incorporated the E6 and E7

open reading frames (ORFs) in their genome [65]. In this study, the authors targeted the

oncogene expression to the ocular lens (through the αA crystallin promoter) and showed

that these genes potentiate cell proliferation and impair cell differentiation. At the same

time, another study reported the development of neuroepithelial tumours in HPV16 E6/E7

transgenic mice that employed the human β-actin promoter to target the expression of

oncogenes [66]. However, these models failed to adequately target the expression of HPV

oncogenes to relevant tissues and were unsuitable for the study of HPV-associated

cancers occurring in patients. Besides these, other in vivo animal models were created

and ameliorated. Thus, mice expressing the early proteins E5, E6 or E7 alone were

created under the control of the keratin-14 (K14) promoter, targeting gene expression to

keratinocytes [67-69]. The targeting of HPV DNA to keratinocytes allows a great similarity

with the multistep process of HPV-associated carcinogenesis that occurs in the clinical

disease in patients. These models allow a better understanding of these proteins’

functions in vivo, which are expected to be similar to their roles in humans. For instance,

E5 transgenic mice treated with estrogen showed that this gene is capable of inducing

cervical cancer alone and, when E6 and E7 are present, these act synergically with E5 to

induce more severe lesions [70]. Additionally, E5 collaborated with the E7 oncogene to

increase the number and size of tumours. This alerts to the importance of the E5

oncogene, which may have a finer role in human cancers than is usually assumed and,

therefore, requires further investigation. In vivo animal models have also been playing a

part in the study of other HPV-associated malignancies like head and neck [71] or anal

[72] neoplasia. With the aid of K14-E6 and -E7 transgenic mice, it has been shown that E7

is the prevailing oncogene in promoting the development of 4-NQO-initiated head and

neck squamous cell carcinoma (HNSCC) [71]. In addition, K14-E6E7 double-transgenic

mice developed more and larger tumours and with superior histopathological severity than

K14-E7 single-transgenic mice. This result highlights the role of E6 which acts in synergy

with E7 to develop HNSCC.

The K14-HPV16 is another transgenic mouse model that has been explored in the

study of inflammation-driven carcinogenesis associated with HPV infection. These mice

incorporate in their genome the entire early region of the human papillomavirus type

16DNA and the expression of these HPV genes is also targeted to basal keratinocytes.

This model is more adequate to study the events of carcinogenesis because it comprises

all the virus oncogenes that control the early stages of the disease whilst the previously

11

described models target oncogene expression to uncharacteristic locations or are

transgenic for only certain oncogenes, thus helping to study the role played by the

respective protein. The K14-HPV16 mouse model was created by Arbeit et al. in 1994 [73]

and further characterized in 1996 [74]. A landmark study demonstrated that chronic

estrogen exposure induces the malignant transformation of squamous epithelium of the

cervix and vagina in transgenic mice [75]. In fact, a cross-species comparison study

showed great similarity in the features of carcinogenesis between human cervical disease

and K14-HPV16 transgenic mice [76]. Both patients and transgenic mice present

histological stages characteristic of squamous carcinogenesis marked by an increase of

VEGF expression and an up-regulation of inflammation and angiogenesis during

squamous carcinogenesis stages. This evidence strengthens the pertinence of this animal

model to study the multistage process of carcinogenesis and the roles of innate and

adaptive immunity. Recently, immunocompromised mice receiving tumour xenografts

have been used as a model to study novel therapeutics, new molecular targets and to

understand the biologic mechanisms of treatment sensitivity and/or resistance [77, 78].

However, these mice do not have a functional immune system and thus cannot mount an

effective immune response against the tumour. In these mice, the tumour growth is

heterotopic, instead of orthotopic, due to its atypical location. Additionally, in this model,

the lesions do not develop through a multi-step process like the ones occurring in HPV-

induced carcinogenesis but appear with a malignant phenotype at time of transplantation.

Therefore, immunocompromised mice xenografts do not seem reliable to be used in the

proposed HPV-associated studies as they do not mimic the carcinogenesis process

occurring in clinical disease. The K14-HPV16 transgenic mouse model should be a fitting

alternative to gain knowledge of these matters.

Table 1: In vitro and in vivo models for HPV research. Abbreviations: CRPV – Cot tontail Rabbit Papillomavirus; CPV-1 – Canine Oral Papillomavirus; BPV – Bovine Pa pillomavirus.

Model Main Characteristics References

In vitro

HeLa, SiHa, CaSki Cell lines obtained from cervical cancer patients, expressing HPV-18 (HeLa) or HPV-16 (SiHa and CaSki).

[79-84]

Organotypic cultures

Proliferating keratinocytes cultured in a dermal-equivalent support; Allows some degree of histologic interpretation; May be used to mimic HPV-induced squamous lesions.

[60, 61]

In vivo

Cottontail Rabbit, Dog, Bovine

Naturally or experimentally infected with CRPV, CPV and BPV, respectively; Allows comparative studies with HPV; Their use as models is limited by ethical and financial edges.

[62]

12

Mice

αA-HPV16-E6/E7 HPV oncogenes are expressed in the mice ocular lenses; Does not affect the animal viability.

[65]

hAc-HPV16-E6/E7 Develop neuroepithelial tumours; Is not possible to define a pre-neoplastic phase; Exhibit rapid malignant progression.

[66]

αHPV18-E6/E7

HPV-18-E6/E7 oncogenes are targeted to the ocular lenses; May be useful to study some factors that play a part in carcinogenesis, namely the roles of p53 and pRB proteins.

[85]

UC-HPV11-E2 The HPV-11-E2 gene is expressed in several cell types; Useful for the study of the E2 protein.

[86]

HPV18-URR, HPV11-URR

The upstream regulatory region of HPV type-18 or -11 is expressed only in epithelial cells; For both types, HPV is active in gland cells which may originate adenocarcinomas.

[87, 88]

NOD-scid -

IL2Rgamma null (NSG), Nude-Fox

n1null

Immunodeficient mice used for human head and neck tumours xenografts; These models skip the carcinogenic process; May be useful for testing new therapeutic strategies.

[77, 78]

K14-HPV16-E5/E6/E7

HPV-16 oncogenes are expressed only in keratinocytes, mimicking the site of clinical disease; These single-transgenic mice are useful to study the role of the respective oncoprotein in HPV-induced carcinogenesis.

[67-69]

K14-HPV18-E7, K14-HPV8-E2

Allow the study of the function of each oncogenic protein from the respective type of HPV.

[89, 90]

K14-HPV8 The HPV-8 early genes are targeted to be expressed in basal keratinocytes; Is appropriate to the study of HPV8-induced carcinogenesis.

[91]

K14-HPV16

The HPV-16 early genes are expressed in basal keratinocytes; Is very useful to reproduce the multi-stage carcinogenic process occurring in human disease; Shares histological features with human lesions; Shares patterns of angiogenesis with human cervical carcinogenesis.

[73, 74, 76]

K14-HPV16 Transgenic Mice as a Model for Inflammati on-

associated Carcinogenesis

In K14-HPV16 transgenic mice, inflammation rises at the sites where HPV is

expressed and progresses along the time while playing a part in carcinogenesis. For that

reason, this in vivo animal model is suitable for studying the features involved in

inflammation-associated carcinogenesis. As so, this model is also adequate to study the

13

effect of anti-inflammatory drugs as possible therapeutic agents to treat inflammation and

its associated malignancies.

The K14-HPV16 mouse model helped to understand that CD4+ T lymphocytes

may promote the development of dysplasia in the skin instead of having an immune

surveillance function [92]. Mice with a genetic deletion of CD4+ T cells presented a

reduced incidence of carcinomas when compared with controls. These results are in

agreement with the events occurring in human patients, where women with cervical

intraepithelial neoplasia (CIN) 3 lesions had a higher frequency of CD4+CD25hi Treg cells

when compared to patients with CIN0 and CIN1/2 [46]. However, not only CD4+ T cells

may contribute to increased incidence of carcinomas. The absence of B lymphocytes

impairs the ability of innate immune cells to infiltrate and become activated in neoplastic

skin [93]. B cells do not infiltrate the tumour site but they secrete immunoglobulins (Ig)

which are deposited in the neoplastic tissue. The reduction in infiltrating innate immune

cells in premalignant skin results in deficient angiogenesis and diminished keratinocytes

hyperproliferative activity. Therefore, carcinogenesis promoted by chronic inflammation

requires the presence of B cells. In addition, also mast cells (MCs) may be important in

malignant progression. The absence of these cells seems to compromise the initial

features of malignant progression in vivo, while its presence is usually associated with

increased angiogenesis [94], therefore suggesting a pro-tumour role of MCs during cancer

progression. Mast cells, among other immune and stromal cells, are an important source

of the matrix metalloproteinase 9 (MMP9) which seems to play a pro-tumour role as it

increases the incidence of carcinomas in HPV-transgenic mice [95]. However, in the

presence of MMP9 the arising tumours are less aggressive than in its absence. MMP9

appears to act as an important mediator of angiogenesis and regulator of epithelial

proliferation and therefore permits the faster tumour progression when compared to

MMP9-deficient mice [96]. Paradoxically, tissue inhibitors of metalloproteinases (TIMPs),

namely TIMP-1, which should oppose the action of MMPs, also appear to enhance

epithelial carcinogenesis [97]. It is reported that the expression of TIMP-1 increases

during the course of malignant progression in vivo promoting carcinogenesis. This

promotion is most likely due to the TIMP-1 capacity to potentiate the proliferation of

keratinocytes. In addition, also the complement system is activated during multi-step

carcinogenesis, albeit its component C3 is not responsible for recruiting immune cells to

the tumour milieu [98]. In fact, extensive regions of IgG deposition were observed in

hyperplastic and dysplastic skin of HPV16 mice which was comparable to HPV16 mice

lacking the complement protein C3. This outcome suggests an antibody-mediated

mechanism, independent of complement activation, to mediate the recruitment of immune

cells during carcinogenesis. A more recent study suggests that the peripheral activation of

14

an antibody-mediated response promotes the development of squamous cell carcinoma

(SCC) by local binding of the Fc γ-receptor (FcγR) on resident and recruited immune cells

[99]. Else, the dominant-negative c-Jun (TAM67) blocks the activation of the transcription

factors AP-1 (activator protein-1) and NF-κB (nuclear factor-kappa B) which are central in

the process of tumour promotion and progression [100]. Using HPV16 transgenic mice, it

has been shown that TAM67 also inhibits the action of the E7 oncogene upon certain

genes that may enhance carcinogenesis [101]. Additionally, the same study also

describes that TAM67 down-regulates the expression of the pro-inflammatory enzyme

cyclooxygenase-2 (COX-2), therefore acting as a tumour suppressor. Furthermore,

hypoxia is a known feature commonly linked to poor prognosis in most cancers. Hypoxia

induces changes in cancer tissues which may lead to inhibition of apoptosis, genomic

instability, angiogenesis, cellular differentiation, invasion, radio-resistance and other

alterations [102] and so, can be used as a prognostic and therapeutic factor. The hypoxic

cellular response is coordinated by the hypoxia-inducible factor-1 (HIF-1). This

transcription factor promotes the expression of vascular endothelial growth factor (VEGF)

which in turn increases angiogenesis, a known feature related with poor prognosis. In fact,

in vitro studies showed that HPV oncoproteins E6 and E7 enhance VEGF expression

through a HIF-1α-dependent mechanism, thus contributing to increased angiogenesis

[103, 104]. This is in agreement with the observed in K14-HPV16 transgenic mice, where

HIF-1 is up-regulated during the stages of carcinogenesis [105], suggesting that HIF-1

promotes local growth and invasion of cervical cancer, similarly to the natural events in

clinical disease [106]. Furthermore, also integrins play an important role in cancer

progression and, especially, in metastasis, as they are mediators of cell adhesion [107].

Also, the reduced expression of α2β1 integrin resulted in diminished progression from

papillomatosis to dysplasia and decreased tumour growth [108]. These results suggest

that the α2β1 integrin is important in regulating and promoting the initial steps of

carcinogenesis. More, cathepsin L – a lysosomal cysteine endoprotease – can influence

tumour progression in vivo [109]. When cathepsin L is absent in K14-HPV16 mice these

have a higher proliferative activity of keratinocytes, faster development of dysplasias and

increased frequency of metastasis when compared to animals in the presence of this

endoprotease [110]. Therefore, these data are suggestive of an anti-tumour role for

cathepsin L during carcinogenesis. In conflict with this conclusion, another study suggests

that cysteine cathepsins, including cathepsin L, promote tumour progression in vivo [111].

This report shows augmented progression from dysplasia to SCC, enhanced cathepsins

expression, reduced apoptosis, increased cell proliferation and promotion of angiogenesis

in K14-HPV16 mice with cystatin C deficiency. Cystatin C is the most abundant of all

cystatins which are endogenous inhibitors of cathepsins. Therefore it seems paradox that

15

both cathepsin L and cystatin C present anti-tumour functions. Else, it has been reported

that neuropilins – transmembrane receptors for the VEGF family of angiogenic proteins

and the class 3 semaphorin family of guidance proteins – are over-expressed in samples

of dysplasia and SCC from K14-HPV16 transgenic mice when compared to controls [112].

Nevertheless, neuropilins are only expressed in differentiated tumour cells. More recently,

the expression levels of the microRNA (miR)-155 were measured in K14-HPV16

transgenic mice in order to understand the possible influence of HPV with this biomarker

[113]. In this study, the HPV-positive mice presented significantly lower levels of miR-155

when compared with HPV-negative controls, suggesting that low levels of miR-155 may

play a role in early phases of HPV-associated carcinogenesis. The same group of

researchers also analyzed the expression of miR-21 in ear and chest skin samples of

HPV16-/- and HPV16+/- mice [114]. Levels of miR-21 expression were higher in less

aggressive lesions (hyperplasia) when compared with more aggressive ones (carcinoma

in situ), which is in agreement with the anti-inflammatory properties of miR-21 and

suggests that higher levels of this molecule may counteract malignant progression.

Modulators of the immune response and their effect over CD8 + T

cells

As seen earlier, the process of carcinogenesis induced by HPV infection is closely

associated with an intricate immune cell infiltrate and its soluble mediators [115]. Some of

these cell populations are commonly linked with tumour regression, as is the case of M1

macrophages, Th1 and cytotoxic T lymphocytes [12]. The latter, CTL, are specialists in

virus-infected and tumour cell destruction and, therefore, represent a key population in

anti-tumour immunity. These cells become activated when viral or tumour antigens are

presented in MHC I molecules together with the required co-stimulatory signals [116, 117].

Upon activation, CD8+ T cells exert their functions either directly, by releasing cytotoxic

granules (rich in perforin and granzymes) or cell-cell contact (e.g. FAS-FASL); or

indirectly, following an IFN-γ-dependent mechanism [118, 119]. In fact, CTL have already

been shown to infiltrate the tumour microenvironment and promote its regression in

several malignancies including HPV-associated cancers [56, 120, 121].

The occurring immune response can suffer alterations in the presence of

immunomodulatory stimuli which can be induced, for instance, by toxins or medical drugs.

Hence, immunomodulators can promote either immunosuppression or immunostimulation

and, therefore, alter the balance between regulatory and effector cells, resulting in

improved or impaired immune defensive mechanisms [122]. For instance, ptaquiloside, a

carcinogenic toxin found in bracken (Pteridium spp.), has some immunomodulatory effects

16

[123]. Bracken toxins are hypothesized to drive the malignant progression of

papillomavirus-induced upper digestive lesions in cattle and human populations, as

remarked by Chang et al. [124]. Ptaquiloside is a chemically unstable compound that has

been shown to induce splenic white pulp atrophy, neutropenia, reduced NK cells activity

and a B-cell lymphoproliferative malignancy [125-128]. However, until now, there are no

reports of an association between ptaquiloside and CD8+ T lymphocytes. In turn,

celecoxib (CXB) is an immunomodulatory drug which specifically inhibits cyclooxygenase-

2 (COX-2). This enzyme plays a key role in the development of an inflammatory response

[129] and is over-expressed in several types of cancer [130-134]. By selectively inhibiting

COX-2, celecoxib diminishes the production of prostaglandins without significantly

impairing the COX-1 isoform [135]. Actually, a few studies have already enlightened the

effect of this selective COX-2 inhibitor over cytotoxic T cells [136-140]. However, whether

celecoxib enhances or decreases CD8+ T lymphocytes infiltration and activity varies

between studies. Still, none of these studies analysed the effect of celecoxib over CTL in

HPV-associated lesions.

17

Chapter 2: Objectives

The purpose of this work was to assess the infiltration and activity of cytotoxic

CD8+ T lymphocytes in HPV-induced skin lesions, using the K14-HPV16 mouse model.

Thus, the work was focused in some more specific aims:

- To perform a kinetic study of the CTL infiltration and activation by comparing

lesions from different-aged mice;

- To analyse the immunomodulatory effect of ptaquiloside over CD8+ T cell

infiltration and activation;

- To analyse the immunomodulatory effect of celecoxib over CD8+ T cell infiltration

and activation.

19

Chapter 3: Kinetics of CD8 + T cells in human

papillomavirus-induced lesions 1

1 The contents of this chapter were adapted from:

- Santos C., Ferreirinha P., Sousa H., Ribeiro J., Bastos M.M.S.M., Faustino-Rocha A.I., Oliveira P.A., Medeiros R., Vilanova M., Gil da Costa R.M. (2015) Kinetics of CD8+ T cells in human papillomavirus-induced lesions – data from K14-HPV16 transgenic mice. Submitted to the journal Tumor Biology with the number TUBI-D-15-04094.

21

Introduction

Human papillomavirus (HPV) is the main etiologic agent of cervical cancer [4], but

can also originate anal, skin and head and neck malignancies [6, 141, 142]. Only a

persistent infection by an oncogenic HPV type (mainly HPV 16 and 18) can induce

malignant transformation. In approximately 90% of HPV infected individuals the host

immune system is able to fight the virus and hinder its dissemination. However, in the

remaining 10%, HPV can effectively evade immune defenses and lead to clinical disease

[2, 3].

K14-HPV16 transgenic mice, created nearly two decades ago [73], are an useful in

vivo animal model for the study of HPV-induced carcinogenesis. This model shares a

number of morphologic and molecular similarities to HPV-related human disease [76],

thus functioning as an excellent replica of the multi-stage process of carcinogenesis.

Targeting of HPV16 oncogenes to keratinocytes by the keratin-14 (K14)

promoter/enhancer is the key characteristic of this model [73].

HPV-induced carcinogenesis is associated with progressively intense chronic

inflammation. Therefore, a great diversity of immune cells and a multiplicity of soluble

mediators can be found within the tumour microenvironment [18]. The inflammatory

infiltrates provide pro- and anti-tumour stimuli, which will favour either the development or

the regression of the lesion [55, 143].

CD8+ T cells are restricted to major histocompatibility complex class I (MHC I)

molecules which can present peptides generated from intracellular viruses and/or tumour

cells [117]. Upon recognizing specific peptides presented on MHC I molecules on the

surface of professional antigen presenting cells, which also display co-stimulatory

molecules, these T cells can differentiate into cytotoxic T lymphocytes (CTL), helped by

cytokine stimuli [116]. CTL are an important part of tumour-specific immunity. They may

eliminate target cells either directly, through the release of lytic granules containing

several enzymes (such as perforin and granzyme) or by the engagement of death

receptors (e.g.: FAS-FASL), and indirectly, following an interferon-γ-dependent

mechanism which leads to cell cycle inhibition, apoptosis and stimulation of macrophage

anti-tumour activity [118, 119].

Thus, using the K14-HPV16 transgenic mouse model, the aim was to examine the

kinetics of CD8+ T cell infiltration in HPV-induced lesions during multi-step carcinogenesis

at different time points. The goal was also pointed to understand whether these cells were

activated by determining the presence of the lysosome-associated membrane protein 1

(LAMP1), also known as CD107a, at the cell surface.

22

Material & Methods

Animals

Generation of K14-HPV16 mice on a FVB/n background has been previously

reported [73]. K14-HPV16 transgenic mice were generously donated by Drs. Jeffrey Arbeit

and Douglas Hanahan (University of California) through the USA National Cancer Institute

Mouse Repository. The animal experiments were approved by the Universidade de Trás-

os-Montes e Alto Douro ethics committee (10/2013) and the Portuguese Veterinary

Directorate (0421/000/000/2014). Animals were maintained and bred according to

Portuguese (Decreto-Lei 113/2013, August 7th) and European (EU Directive 2010/63/EU)

legislation, under controlled conditions of temperature (23 ± 2 ºC), light-dark cycle (12h

light/12h dark) and relative humidity (50 ± 10 %), using hardwood bedding. Food and

water were provided ad libitum.

Mice genotyping

Animals were genotyped at weaning, using tail tip samples as described previously

[113, 114]. Briefly, nucleic acids were extracted and DNA quality and purity were

assessed. HPV16-E6 and -E2 genes were amplified to confirm the presence of HPV DNA

and mouse β-globin was used as control. Amplicons lengths were confirmed by agarose

gel electrophoresis. Only hemizygous females were used for the transgenic mouse groups

in the experiment.

Study Design

Ten wild-type (WT) (HPV16-/-, Group 1) and twelve transgenic (HPV16+/-, Group 2)

female mice were euthanized at 24-26 weeks of age. Latter, six HPV16-/- (Group 3) and

six HPV16+/- (Group 4) female mice were sacrificed when 28-30 weeks-old. The mice were

humanely sacrificed by intraperitoneal pentobarbital overdose, followed by exsanguination

through cardiac puncture. Chest skin samples (approximately 4 cm2) were collected for

cell isolation and flow cytometry analysis. Matched samples were collected for histological

examination.

Histology

Skin samples were fixated in 10% neutral buffered formalin for 48h. Samples were

dehydrated through graded alcohols and xylene and paraffin-embedded in an automatic

STP 120 processor (Micron, Boise, ID). 2 µm-thick sections were stained with

haematoxylin-eosin (H&E) for histological examination on light microscopy. Skin samples

23

were classified as normal skin, epidermal hyperplasia or epidermal dysplasia. Minor

dysplastic foci on a hyperplastic background were also recorded.

Preparation of Single-Cell Suspensions

Chest skin samples were cleaned from excessive blood vessels and fat tissue and

cut into small pieces. These fragments were incubated for 2 hours with 125 U/ml type I

collagenase (Gibco, Life Technologies, Paisley, UK) in RPMI-1640 medium

complemented with 1% glutamine, 1% penicillin-streptomycin-amphotericin B, 1% HEPES

buffer (all from Sigma, St. Louis, MO) and 10% foetal bovine serum (BioWest, Nuaillé,

France) at 37 ºC and 150 rpm in a 3031 orbital incubator (GFL, Burgwedel, Germany).

The resulting cell suspension was then filtered and centrifuged at 300 � for 10 min at 4 ºC.

Cells were then ressuspended in phosphate buffered saline containing 1% bovine serum

albumin and 20 mM sodium azide followed by extracellular staining.

Immunophenotyping

Following cell isolation, the surface phenotype of the collected cells was assessed

by flow cytometry using specific monoclonal antibodies (mAb). To prevent non-specific

antibody binding, cells were incubated with anti-mouse CD16/CD32 mAb for FcγR

blocking. This was followed by incubation with anti-CD8 mAb phycoerythrin-cychrome 5-

conjugate (clone 53-6.7, BD Biosciences, San Diego, CA) and anti-CD107a mAb

phycoerythrin-conjugate (clone eBio1d4b, eBioscience, San Diego, CA). Following

extracellular staining, the cells were washed, fixed in 2% formaldehyde and washed with

phosphate buffered saline containing 1% bovine serum albumin and 20 mM sodium azide.

Antibody-labelled cells were analysed in an EPICS XL flow cytometer using the

EXPO32ADC software (Beckman Coulter, Miami, FL). The collected data files were

analysed using the FlowJo software v10.0.7 (FLOWJO, LLC, Ashland, OR).

Statistical Analysis

Statistical analyses were executed in the GraphPad software (version 6.0,

GraphPad Software, Inc. La Jolla, CA). Statistical analysis between group pairs was

performed using the Mann-Whitney test.

24

Results

Transgenic Mice Show Epidermal Hyperplasia and Dysp lasia

Analysis of skin samples showed that the totality of WT mice (groups 1 and 3)

presented normal skin histology (Fig. 2a) whilst all transgenic mice presented skin lesions.

In all 12 mice composing group 2 it was possible to observe simple to papillary, diffuse,

variably severe epidermal hyperplasia and papillomatosis with orthokeratotic

hyperkeratosis (Fig. 2b). Inflammation was mild, with a few macrophages, lymphocytes

and mast cells present in the superficial dermis. Also in this group, 2 animals (16.7%)

presented small epidermal dysplastic foci. In group 4, 3 animals (50.0%) presented diffuse

epidermal dysplasia (Fig. 2c); other 3 (50.0%) showed multifocal epidermal dysplasia in a

hyperplastic background. Sub-epidermal angiogenesis and dermal inflammatory infiltrates

were prominent, showing numerous mixed mononuclear leukocytes and neutrophils.

Fig. 2: Histopathological changes induced by HPV16 oncogene s in FVB/n mice, H&E. a – WT animal; Normal skin histology, �� . b – 24-26 week-old HPV16 +/- animal; Epidermal hyperplasia extending to the fol licular infundibulum and isthmus, �� . c – 28-30 week-old HPV16 +/- animal; Epidermal dysplasia, �� . Note enhanced sub-epidermal inflammatory cell infiltration.

Increased CD8 + T lymphocytes numbers and activation in HPV16 +/- mice

In order to assess the presence of CD8+ T cells in HPV-associated lesions,

lymphoid cells were isolated from chest skin tissue and analysed by flow cytometry (Fig.

3a). As shown in Fig. 3b, chest skin samples from HPV16+/- mice (groups 2 and 4)

presented a significantly higher percentage of CD8+ T cells when compared with those of

WT animals (groups 1 and 3). Although the percentage of skin CD8+ T cells was slightly

higher in group 4 than in group 2, it did not reach statistical significant difference.

In order to determine if the CD8+ T cells found in the skin of HPV16+/-

mice

presented evidence of cytotoxic activity, the surface expression of CD107a was

evaluated. In CD8+ T lymphocytes CD107a reaches the cell surface when lytic granules

suffer exocytosis, thus exposing its membrane proteins [144]. Therefore, this lysosome-

associated membrane protein is a commonly used surrogate marker of CTL degranulation

[145]. As shown in Fig. 3c, the percentage of CD107a+CD8+ T cells in chest skin samples

25

of mice from groups 2 and 4 were found significantly higher when compared with

respective controls of groups 1 and 3. This indicates that in the HPV16+/- mice skin

infiltrating CTL released cytotoxic granules. Moreover, in group 4 mice, 100% of which

show multifocal or diffuse dysplasia, a markedly higher percentage of CD8+ T cells

express CD107a (P � 0.001) than in group 2 mice, of which only 16.7% show focal

dysplastic lesions. This result shows a positive correlation in the proportions of activated

CD8+ T cells and lesion severity.

Fig. 3: Percentage of CD8 + and CD8+CD107a+ T cells within total lymphoid-gated cells obtained from chest skin samples from WT and K14-HPV16 transgenic mice. a – Representative analysis of the gating strategy empl oyed. Numbers within graphs correspond to the percentage of the gated population. b – Percentages of CD8 + T cells and c – percentages of CD8 +CD107a+ T cells in gated CD8 + T cells were determined by flow cytometry after sk in tissue collection and digestion with collagenase. Chest sk in samples were collected from 24-26 weeks-old HPV -/- and HPV+/- mice (Groups 1 and 2, respectively) and 28-30 week s-old HPV -/- and HPV+/- mice (Groups 3 and 4, respectively). Group 1, n = 10; Group 2, n = 12; Gr oup 3, n = 6; Group 4, n = 6. Each dot represents a n individual animal. Bars represent the mean value in each group . ** P � 0.01; *** P � 0.001.

26

Discussion

HPV is considered to be the cause of virtually all cases of cervical cancer [146],

therefore playing a central role in carcinogenesis. Despite the continuous progress in the

understanding of the immune response against HPV and the development of effective

vaccines, much is still ignored about the cell-mediated response against HPV-induced

lesions. HPV-mediated carcinogenesis is commonly associated with chronic inflammation

[18]. Nevertheless, the complex interactions within the inflammatory infiltrate remain not

fully understood [39].

The K14-HPV16 transgenic mouse model appears as an appropriate in vivo

animal model to improve the knowledge on HPV-induced carcinogenesis and elicited

immune response. Besides cervical cancers, these mice develop aggressive skin lesions

with greater incidence in the ear and the chest [74]. As expected, it was possible to detect

a higher incidence of aggressive lesions (epidermal dysplasia) in older transgenic mice

than in younger ones, which presented mostly hyperplastic lesions. The development of

dysplastic lesions was accompanied by a dramatic intensification of sub-epidermal

inflammation. As this process of carcinogenesis is associated with progressive chronic

inflammation, this model will allow the study of the intervening immune effectors.

CD8+ CTL are important players in tumour-specific immunity [147]. When CD8+

CTL recognize their targets they release lytic granules containing a variety of enzymes

(perforin, granzyme and other) which allow the killing of virus-infected and tumour cells

[118]. During exocytosis, CD107a reaches the cell surface as the membrane of lysosomes

disrupts, thus helping to determine whether the CD8+ T cells are activated [145].

In this work, it was possible to observe that CD8+ T cells heavily infiltrate HPV-

induced chest skin lesions in K14-HPV16 transgenic female mice. This data is in

agreement with previous studies which described the presence of CD8+ T cells in HPV-

induced cervical and skin lesions in human patients [43, 148]. According to one of these

studies, the presence of CD8+ T cells in cervical tissue was associated with tumour

regression [148]. The results presented here suggest a trend towards the increase of

infiltrating CD8+ T cells in chest skin samples of 28-30 weeks-old mice when compared to

24-26 week-old animals. Although indicating that CD8+ T lymphocytes migrate in greater

number towards more severe lesions than to less aggressive ones, these results should

be further confirmed. Nevertheless, this work’s data clearly show that a significant

proportion of the infiltrating CTL are activated and degranulate. Whether these cells were

activated at the lesion site or at regional lymph nodes would be an appealing point to

address hereafter. CD8+ T lymphocytes isolated from older animals present a high degree

of activation when compared to that isolated from younger mice. This suggests that more

27

CD8+ T cells become activated in response to a more severe stimulus than when

confronted with a less aggressive lesion. However, despite the increase in number of

cytotoxic cells and its enhanced activation, the lesions still tend to progress to a poor

phenotype with aging. Low tumour infiltration by activated CTL has been associated with

the limiting efficacy of the immune response in eliminating established tumours [149]. The

results reported herein indicate that however important, CTL recruitment and

degranulation into lesion areas do not suffice to prevent progressive carcinogenesis in the

K14-HPV16 transgenic mice. Many immunoevasive strategies have been identified

preventing the effectiveness of tumour immunity mediated by CTL [150]. However, as a

high proportion of skin infiltrating CD8+ T cells showed evidence of degranulation in the

older K14-HPV16 transgenic mice, it would be worth exploring whether the transgenic

cells might present an intrinsic resistance to cytotoxic mechanisms dependent on CTL

degranulation.

Conclusion

The results presented here clearly show that CD8+ T cells infiltrate HPV-induced

lesions, but indicate their activation is not enough to stop malignant progression in this

model. These results also support the use of K14-HPV16 mice as an adequate model to

study the host immune response associated with HPV-induced lesions and help

developing immunotherapeutic strategies that could prevent carcinogenesis.

29

Chapter 4: Ptaquiloside inhibits tumour-infiltratin g CD8+

T cells in HPV-transgenic mice

31

Introduction

Papillomavirus cause benign and malignant lesions in many wild and domestic

animal species, as well as in human populations. Human papillomavirus (HPV) is the

source of several diseases, including many anogenital and some upper digestive cancers

[151]. The growing incidence of HPV-positive oropharyngeal cancer is now of particular

concern [152]. Several bovine papillomavirus (BPV) types, such as BPV1, BPV2 and

BPV4 are associated with cutaneous and upper digestive papillomatosis, as well as with

abortive urinary bladder infections [63]. Interestingly, digestive tract cancers induced by

HPV and BPV share many common features and, possibly, some environmental co-

factors [124].

A persistent infection by an oncogenic viral type is mandatory for malignant

progression to take place [3]. Viral persistence is determined by a complex interplay

between the host immune system and the virus ability to evade it. In this context,

environmental immunosuppressants are likely to play an important role.

Bracken (Pteridium spp.), is a fern belonging to the Pteridaceae family with a

worldwide distribution [123]. It is known for its ability to facilitate BPV persistence in

infected cattle, promoting the malignant transformation of upper digestive papillomas into

squamous cell carcinomas [153]. Importantly, exposure to bracken and its toxins has also

been associated with an increased risk of developing upper digestive malignancies in

human populations (reviewed in [123]). Ptaquiloside, the main bracken toxin, is an

unstable nor-sesquiterpene glycoside of the illudane family, with important immunotoxic

properties. It has the ability to induce neutropenia and inactivate natural killer (NK) cells,

which play an important role in innate immunity, by killing virus-infected and neoplastic

cells [126, 127]. Therefore, it might be hypothesized that these immunosuppressive

effects could facilitate viral persistence and the progression of early-stage papillomavirus-

induced lesions.

Cytotoxic CD8+ T lymphocytes (CTL) are another cell population involved in host

immunity to virus-infected and neoplastic cells as the main responsible for cytotoxic

functions in an adaptive immune response. CTL target virus-infected and tumour cells via

antigen presentation by Major Histocompatibility Complex class I (MHC I) molecules [117],

in the presence of other co-stimulatory signals [116]. These cells may directly induce the

killing of target cells, by releasing lytic granules or through death receptor engagement

(e.g. FAS-FASL) or indirectly, by producing IFN-γ, which leads to multiple anti-tumour

activities [118, 119]. Several HPV and BPV types (including HPV16 and BPV1 and 4) are

able to down-regulate MHC I expression through their E5 oncoprotein, as an immune

evasion strategy [154]. However, recent findings confirm that, even under conditions of

32

low MHC I expression, CD8+ T cells play a decisive role in fighting HPV-induced

oropharyngeal cancers [155].

In the present work, we hypothesize that ptaquiloside, the bracken toxin, exerts its

immunosuppressive effect by counteracting the action of CD8+ T cells against

papillomavirus-induced lesions. This would facilitate viral persistence and the progression

of early HPV- and BPV-induced lesions, posing major risks for human and animal health.

In order to test that hypothesis, we employed HPV16-transgenic mice and analysed the

effect of ptaquiloside on the population of skin infiltrating CD8+ T cells.

Material & Methods

Mice

Construction of K14/HPV16 mice on a FVB/n background has been previously

reported [73]. These animals develop characteristic multi-stage cutaneous and uterine

cervical carcinogenesis, and were generously donated by Drs. Jeffrey Arbeit and Douglas

Hanahan (University of California) through the USA National Cancer Institute Mouse

Repository. The animal experiments were approved by the Universidade de Trás-os-

Montes e Alto Douro ethics committee (10/2013) and the Portuguese Veterinary

Directorate (0421/000/000/2014). Animals were maintained and bred according to

Portuguese (Decreto-Lei 113, August 7th) and European (EU Directive 2010/63/EU)

legislation, under controlled conditions of temperature (23 ± 2 ºC), light-dark cycle (12h

light/12h dark) and relative humidity (50 ± 10 %), using hardwood bedding. Food and

water were provided ad libitum.

Mice genotyping



assessed. HPV16-E6 and -E2 genes were amplified to confirm the integration of HPV

DNA into the mouse genome and a fragment of mouse β-globin was also amplified to

confirm the quality of the extracted DNA. Lengths of the fragments were confirmed by

agarose gel electrophoresis. Only hemizygous female mice were used for the transgenic

mice groups in the experiment.

Ptaquiloside isolation

Ptaquiloside was isolated from bracken as previously described [156] with minor

modifications. Briefly, 1000 g (dried weight) bracken crosiers were harvested at Arcos de

Valdevez, Portugal, 41º 49´ 12´´ N, 8º 24´ 11´´ W) and a sample was deposited at the

33

Universidade de Trás-os-Montes e Alto Douro herbarium (reference no. 18248). Bracken

was blended in distilled water (10 L), stirred at room temperature for 1 hour and the

extract was adsorbed on 3 L of XAD-2 resin (Supelco, Sigma, St. Louis, MO). The resin

was eluted with methanol (10 L) and the methanol extract was concentrated, dissolved in

water (400 ml) and extracted with butanol (5�500 ml). The butanol extract was

chromatographed on silica gel (Merck, Kenilworth, NJ). Fractions containing ptaquiloside

were separated twice on octadecyl-sylane silica gel (Fujy-Silysia, Kasugai Aichi, Japan)

using methanol-water mixtures to obtain pure ptaquiloside. The compound was

distinguished from other closely-related bracken illudane toxins on the basis of its

characteristic 1H and 13C nuclear magnetic resonance (NMR) signals [157] using an

Avance III 400 MHz spectrometer (Brucker, Billerica, MA). Aliquots were prepared for

each experimental week (7.5 mg), freeze-dried and kept at -20ºC until use.

Study Design

Thirty transgenic (HPV16+/-) and 15 wild-type (WT, HPV16-/-) female 18-20 weeks-

old mice, showing diffuse cutaneous crusting and papillomatosis were employed. The

animals were separated into three experimental groups: group 1 (n = 15, HPV16-/- mice),

group 2 (n = 15, HPV+/- untreated mice) and group 3 (n = 15, HPV16+/- mice treated orally

with 0.5 mg ptaquiloside per week, for 10 consecutive weeks). The experimental animals

were monitored daily for signs of stress or disease. All surviving mice were euthanized at

28-30 weeks of age by an intraperitoneal pentobarbital overdose, followed by cardiac

puncture and exsanguination. Chest skin samples (approximately 4 cm2) were collected

for flow cytometry analysis. Matched samples were collected for histological examination.

Ptaquiloside administration and toxicity

Each week, a 7.5 mg ptaquiloside aliquot was dissolved in 300 µl ethanol (25

mg/mL) and the individual 0.5 mg dose (20 µl ethanol) was added to a standard wheat

cookie (Vieira, Vila Nova de Famalicão, Portugal) fragment weighting ca. 100 mg. Each

dosed fragment was allowed to dry at room temperature for 10 minutes and individually

administered to a group 3 animal, in an individual empty cage; the ingestion was visually

monitored. In order to confirm that ptaquiloside was indeed active at the administered

dosage, we looked for a lymphoid malignancy [128]. Thus, histological analysis of kidney

samples was used to confirm neoplastic lymphoid cell infiltration in this organ.

34

Skin histology

Skin samples were fixated in 10% neutral buffered formalin. Samples were

dehydrated through graded alcohols and xylene and, paraffin embedded in an automatic

STP 120 processor (Micron, Boise, ID). 2 µm-thick sections were stained with

haematoxylin-eosin (H&E) for histological examination on light microscopy. Skin samples

were classified as normal skin, epidermal hyperplasia, multifocal epidermal dysplasia in a

hyperplastic background and diffuse epidermal dysplasia.

Isolation of a Single-Cell Suspension

Chest skin samples were cleaned from excessive blood vessels and fat tissue and

cut into small pieces. This fragments were incubated for 2 hours with 125 U/ml of type I



buffer (all from Sigma) and 10% foetal bovine serum (BioWest, Nuaillé, France) at 37 ºC

and 150 rpm in a 3031 shaking incubator (GFL, Burgwedel, Germany). The resulting cell

suspension was then filtered and centrifuged at 300 � for 10 min at 4 ºC. Cells were then

ressuspended in phosphate buffered saline containing 1% bovine serum albumin and 20

mM sodium azide followed by extracellular staining.

Flow Cytometry Analysis

Following cell isolation, the cellular immune phenotype was assessed by flow

cytometry using monoclonal antibodies (mAb). Non-specific antibody binding was

prevented by incubating cells with anti-mouse CD16/CD32 mAb for FcγR blocking. This

was followed by incubation with anti-CD8 phycoerythrin-cychrome 5-conjugate (clone 53-

6.7, BD Biosciences, San Diego, CA), anti-CD107a (LAMP1) phycoerythrin-conjugate

(clone eBio1d4b) and anti-CD44 phycoerythrin-cychrome 7-conjugate (clone IM7) mAb

(both from eBioscience, San Diego, CA). Following extracellular staining, the cells were

washed, fixed in 2% formaldehyde and washed with phosphate buffered saline containing

1% bovine serum albumin and 20 mM sodium azide. Antibody-labelled cells were

analysed in an EPICS XL flow cytometer using the EXPO32ADC software (Beckman

Coulter, Miami, FL). The collected data files were analysed using the FlowJo software

v10.0.7 (FLOWJO, LLC, Ashland, OR).


Flow cytometry statistical analyses were executed in the GraphPad software

(version 6.0, GraphPad Software, Inc. La Jolla, CA). In column and dot graphs, each point

is representative of an individual mouse and bars represent the mean value for the

35

respective group. Analysis between group pairs was performed using the Mann-Whitney

test. Kaplan-Meier survival analysis coupled with a log rank test was performed using the

PASW Statistics software (version 18, IBM® SPSS®, Quarry Bay, Hong Kong).

Results

General findings

Ptaquiloside was isolated at a 0.01% yield from bracken, as previously reported

[128]. The compound's structure (Fig. 4a) was confirmed using NMR analysis and no

other illudane glycoside was detected (Fig. 4b, c). Transgenic animals showed

characteristic diffuse cutaneous hyperkeratosis and erythema, together with variably

intense pruritus, while WT mice showed normal skin. All mice in groups 1 and 2 survived

the 10 weeks experimental period. Ten mice (66.7%) from group 3 succumbed before the

end of the study (Fig. 4d). Histological analysis of kidney samples showed normal

histology in WT and untreated HPV+/- animals (Fig. 4e) and, moderate to severe,

multifocal, perivascular infiltration of lymphoblastic cells, showing highly pleomorphic

nuclei and up to 3 mitotic figures per high-power field in 100% of ptaquiloside-treated

animals (group 3) (Fig. 4f).

36

Fig. 4: Ptaquiloside and its leukaemogenic effect in K14-HP V16 mice. a – ptaquiloside's structural formula. b and c – partial 2D NMR-HSQC spectra (obtained in CD3OD) for ptaquiloside. Note the correlations between ca rbons and their corresponding hydrogens in the glucose residu e (b). Note in particular the correlations between carbons 12 and 13 at 5.87 ppm and 10.86 ppm respectively, and their corresponding hydrogens in the cyclopropylide ne ring (c). d – Kaplan-Meier survival analysis. HPV +/- mice treated with ptaquiloside show significantly reduced survival ( P � 0.001) compared with untreated HPV -/- or HPV+/- animals. e – Untreated HPV +/- animal (group 2), showing normal kidney histology; H&E 200 �. f – Ptaquiloside-exposed HPV +/- animal (group 3) showing typical ptaquiloside-associated perivascular infiltration by leukaemic l ymphocytes. Note marked nuclear pleomorphism and mi totic figures; H&E 200 �.

37

Cutaneous lesions

The histological analysis showed that all group 1 mice (100.0%) presented normal

skin histology (Fig. 5a) whilst all transgenic mice (groups 2 and 3) presented skin lesions.

In group 2, 3 animals (50.0%) presented diffuse epidermal dysplasia (Fig. 5b); other 3

(50.0%) showed epidermal hyperplasia with multifocal dysplasia. Sub-epidermal

angiogenesis and dermal inflammatory infiltrates were prominent in dysplastic lesions,

showing numerous mixed mononuclear leukocytes and neutrophils. In group 3, the totality

of mice (100.0%) showed diffuse epidermal dysplasia (Fig. 5c).

Fig. 5: Histopathological changes induced by HPV16 oncogene s in FVB/n mice, H&E. a – group 1 animal. Normal skin histology, 400 �. b – group 2 animal. Epidermal hyperplasia extendi ng to the follicular infundibulum, 400 �. c – group 3 animal. Epidermal dysplasia, 400 �. Note loss of keratinocytic polarity and different iation.

CD8+ T lymphocytes are present in ptaquiloside-treated and untreated

animals

In order to determine the proportions of CD8+ T cells present in chest skin with or

without HPV-associated lesions, samples were collected and cells isolated, followed by

flow cytometry analysis (Fig. 6a). As shown in Fig. 6b, group 2 and 3 (HPV+/- and

ptaquiloside-treated HPV+/-, respectively) mice showed significantly higher percentages of

CD8+ T cells compared with HPV-/- mice (P � 0.05). The percentage of CD8+ T cells in

chest skin was not significantly different between ptaquiloside-treated and untreated

HPV16+/- mice (groups 3 and 2, respectively).

38

Fig. 6: Percentage of CD8 + T cells within total lymphoid-gated cells obtained from chest skin samples from WT and K14-HPV16 transgenic mice. a – Representative analy sis of the gating strategy utilized. The number wit hin the graph corresponds to the percentage of the gated po pulation. b – Percentages of CD8 + T cells were determined by flow cytometry after skin tissue collection and enz ymatic digestion. Chest skin samples were collected from 28-30 weeks-old HPV -/- (Group 1), HPV +/- mice (Group 2) and ptaquiloside-treated (PTQ) HPV +/- mice (Group 3). Group 1, n = 5; Group 2, n = 5; Group 3, n = 5. Each dot represe nts an individual animal. Bars represent the mean v alue in each group. * P � 0.05.

Ptaquiloside decreases the number of CD8 +CD107a+ T cells in HPV16 +/- mice

Next, CD8+ T cells found in skin samples were analysed with the objective to

determine if they were actively degranulating, which indicates ongoing cytotoxic activity.

For that purpose, flow cytometry analysis was performed to assess the expression of

surface CD107a in these cells (Fig. 7a). A significantly higher percentage of

CD8+CD107a+ T lymphocytes was observed in HPV+/- mice as compared with HPV-/-

animals (P � 0.01) (Fig. 7b). Additionally, HPV+/- mice exposed to ptaquiloside also

presented a higher percentage of CD8+CD107a+ T lymphocytes when compared to WT

animals, but this was significantly reduced when compared to untreated HPV+/- mice (P �

0.05).

39

Fig. 7: Percentage of CD8 +CD107a+ T cells within total CD8 + T cells obtained from chest skin samples from WT a nd K14-HPV16 transgenic mice. a – Representative analy sis of the gating strategy utilized. Numbers within graphs correspond to the percentage of the gated populatio n. b – Percentages of CD8 +CD107a+ T cells in gated CD8 + T cells were determined by flow cytometry after skin tissue collection and enzymatic digestion. Chest sk in samples were collected from 28-30 weeks-old HPV -/- (Group 1), HPV +/- mice (Group 2) and ptaquiloside-treated (PTQ) HPV +/- mice (Group 3). Group 1, n = 5; Group 2, n = 5; Gro up 3, n = 5. Each dot represents an individual anim al. Bars represent the mean value in each group. * P �0.05; ** P � 0.01.

Reduced number of CD8 +CD44+ T cells in ptaquiloside-treated transgenic

animals Having determined that CD8+ T cells presented a cell surface phenotype

associated with degranulation, expression of CD44, a marker indicating a memory cell

phenotype [158], was also assessed. As shown in Fig. 8 (a and b), a significantly higher

percentage of CD8+CD44+ T lymphocytes was found in HPV+/- mice as compared with

HPV-/- animals (P � 0.01). Ptaquiloside-treated HPV+/- mice presented a significantly

reduced percentage of CD8+CD44+ T lymphocytes compared with untreated HPV+/- mice

(P � 0.05). In fact, group 3 did not show statistically different values to group 1 (P � 0.05).

40

Fig. 8: Percentage of CD8 +CD44+ T cells within total CD8 + T cells obtained from chest skin samples from WT a nd K14-HPV16 transgenic mice. a – Representative analy sis of the gating strategy utilized. Numbers within graphs correspond to the percentage of the gated populatio n. b – Percentages of CD8 +CD44+ T cells in gated CD8 + T cells were determined by flow cytometry after skin tissue collection and enzymatic digestion. Chest skin sam ples were collected from 28-30 weeks-old HPV -/- (Group 1), HPV +/- mice (Group 2) and ptaquiloside-treated (PTQ) HPV +/- mice (Group 3). Group 1, n = 5; Group 2, n = 5; Group 3, n = 5. Each dot represents an individual animal. B ars represent the mean value in each group. * P � 0.05;** P � 0.01.

Discussion

CD8+ T lymphocytes are important effectors of the cell-mediated immune

response, especially against virus-infected or tumour cells [117]. Upon activation these

cells may degranulate, releasing a set of lytic enzymes capable of destroying target cells

[159]. CD107a is a lysosome-associated membrane protein which can be used as

surrogate marker of degranulation [145]. In CD8+ T lymphocytes, CD107a reaches the cell

surface when lytic granules are exocytosed, thus exposing its membrane proteins [144].

As CD8+ T cells need to be activated in order to release cytotoxic granules, the presence

of CD107a at the cell surface is useful to determine their activation status [145].

Additionally, CD8+ T cells may also acquire a memory phenotype, allowing a quicker recall

response, as memory T cells can be more easily activated than naive T cells [159]. CD44

is a cell-surface glycoprotein and is currently considered the best marker to identify

memory CD8+ T cells [158]. Furthermore, CD44 is thought to act as a regulator of the

motility of CTL in the tumour microenvironment [160].

41

Although many papillomavirus (including some oncogenic HPV and BPV types)

down-regulate MHC I expression as a strategy to evade immune surveillance mediated by

CTL [154], these cells still play a major role against papillomavirus-induced lesions,

namely in HPV-induced oropharyngeal cancers [155]. Evading the host immune response

is critical for maintaining a long-term infection, allowing tumour progression. In this

context, environmental (e.g. dietary) immunosuppressant products such as bracken and

its toxin, ptaquiloside, may play an important role.

In this work, as expected, WT mice had no HPV-associated lesions, whilst age-

matched K14-HPV16 transgenic mice showed hyperplastic lesions with dysplastic foci or

diffuse dysplasia. It is possible that the diffuse dysplastic lesions observed in ptaquiloside-

treated animals represent a more aggressive stage in multi-step carcinogenesis compared

with the often focal dysplasia observed in untreated animals. However, the small number

of animals that survived the experimental period and the absence of obviously invasive

lesions do not allow us to firmly conclude that ptaquiloside enhanced tumour progression.

The increasing aggressiveness of dysplastic versus hyperplastic lesions was

accompanied by an increased bulk of infiltrating immune cells.

These results indicate that HPV-induced hyperplastic and dysplastic skin lesions in

K14-HPV16 transgenic mice show increased infiltration of CD8+ T cells when compared to

normal skin from age-matched WT mice. Ptaquiloside reduced the CD8+ T cell infiltration,

although only slightly (P > 0.05). Nevertheless, ptaquiloside did induce a significant

reduction in CD107a+ and CD44+ CD8+ T cells.

Altogether, these data show that cytotoxic T cells migrate to the site of HPV-

induced lesions, are activated to degranulate and acquire a memory-phenotype and, that

ptaquiloside is capable of impairing the function of these cells, explicitly by inhibiting

cellular activation and memory phenotype acquisition. The molecular mechanism behind

this impairment should be an alluring topic for future research.

Grazing animals and human populations worldwide are easily exposed to bracken

and its illudane toxins, including ptaquiloside and other related compounds [123]. Bracken

is an abundant weed, especially in poorer pastures, and ptaquiloside accumulates in

bovine and ovine milk and meat [161-163], besides contaminating underground waters

[164]. Moreover, some human populations in different countries consume bracken shoots

(known as croziers, broto de samambaia in Portuguese or warabi in Japanese) as part of

their daily diet [123]. In China, the production of dry Pteridium aquilinum var. latiusculum

for food uses (locally known as juecai) was estimated to involve approximately 1000

companies in a business worth 300 million USD per annum [165]. This is considered the

most widely-consumed fern in China, with an estimated annual production of 1200 tons in

the Zhouzhou county, province of Hunan. Bracken and ptaquiloside are well-known

42

carcinogens [128, 166-169] but also show important immunotoxic properties. The present

ptaquiloside yield is in agreement with other previous studies using samples from the

same area [128]. Ptaquiloside extreme instability makes its isolation, manipulation and

administration very challenging, as it easily originates the inactive pterosin B [157].

However, the typical toxicities and dramatic mortality rate now induced leave no doubts as

to its activity. The dosage now employed was similar to the one used intraperitoneally on

CD-1 mice [128]. However, the transgenic K14-HPV16 mouse strain shows added frailty

due to its characteristic lesions, and this is likely to explain the higher mortality now

observed.

Bracken induces other immunosuppressive effects involving innate immune cells.

These include neutropenia and the down-regulation of NK cell activity [127]. This second

effect was recently proposed to contribute to urethane-induced lung carcinogenesis [170].

However, due to ptaquiloside scarcity and instability, studies on bracken toxicology

frequently resort to complex and poorly characterized plant extracts, thereby

compromising the study's ability to draw significant conclusions. The present study is the

first to address the immunosuppressive effects of ptaquiloside in a relevant model of

papillomavirus-induced cancer.

Over the years, the K14-HPV16 transgenic mouse model emerged as a valuable in

vivo mock-up for the study of HPV-induced multi-stage carcinogenesis [73, 74].

Importantly, the expression of viral genes is targeted to basal keratinocytes, the cellular

type affected by natural papillomavirus infections. The high histological resemblance

between the lesions taking place in transgenic mice and the ones occurring in human

patients support the utility of this animal model [76]. Furthermore, K14-HPV16 mice are

useful to study the HPV-associated immune response that accompanies carcinogenesis

[93, 113, 114].

The knowledge on the immune response surrounding papillomavirus-induced

carcinogenesis is in constant growth but the cell-mediated immune response against

neoplastic and pre-neoplastic lesions remains poorly understood. This lack of knowledge

may be holding back the development of more effective therapeutic strategies, including

therapeutic vaccines.

Conclusion

The results confirm the role of ptaquiloside as an immunosuppressive toxin,

capable of enhancing the immune evasion strategies characteristic of papillomavirus-

transformed cells. Ptaquiloside reduces CD8+ T cell infiltration of papillomavirus-induced

lesions, as well as the effector and memory functions of these cells, as shown by a

43

reduced expression of CD107a and CD44. These findings are of particular concern in the

context of oropharyngeal high-risk papillomavirus infections, where dietary toxicants like

ptaquiloside may promote tumour progression and aggressiveness.

45

Chapter 5: Celecoxib promotes degranulation of CD8 + T

cells in HPV-induced lesions of transgenic mice 2

2 The contents of this chapter were adapted from:

- Santos C., Neto T., Ferrerinha P., Sousa H., Ribeiro J., Bastos M.M.S.M., Oliveira P.A., Medeiros R., Vilanova M., Gil da Costa R.M. (2015) Celecoxib promotes degranulation of CD8+ T cells in HPV-induced lesions of transgenic mice. Submitted to the journal Antiviral Research with the number AVR-D-16-00002.

47

Introduction

Human papillomavirus (HPV) has been described as an etiologic factor of several

diseases, such as cervical and other anogenital cancers, and a subset of HPV-positive

head and neck cancers [4, 6, 141, 142]. Oropharyngeal cancers have lately drawn

considerable attention, given their growing incidence [152]. In order for a HPV-associated

neoplasm to develop, a persistent infection by an oncogenic HPV type (mostly types 16

and 18) is mandatory [3]. Only in a minority of cases, in which the immune system fails to

eradicate the virus, will the infection progress to cancer [2].

Cytotoxic T lymphocytes (CTL), differentiated from CD8+ T cells, play an important

role in the complex immune response which accompanies HPV-induced carcinogenesis

[11, 148]. CD8+ T cells are recruited to the lesions microenvironment by chemokines

released by stromal and/or innate immune cells on site [149]. In the presence of activator

stimuli, differentiated CTL can specifically target virus-infected and tumour cells, which are

eliminated either through the release of lytic granules, engagement of death receptors

(e.g. FAS-FASL or TRAIL) or following interferon-γ-dependent mechanisms [116-118]. In

particular, CTL are critical to control cervical lesions and were recently demonstrated to

drive the immune response in patients following the administration of an experimental

therapeutic vaccine [171]. Recent studies have also shown the number of CD8+ T cells to

be an important independent prognostic marker in HPV-positive head and neck cancer

patients, while high CD8+ T cell levels correlate with a better prognosis, enhancing overall

survival as well as progression-free survival [155, 172, 173]. Understanding and

enhancing CTL function in patients with HPV-induced malignancies seems, therefore, a

priority for cancer therapy.

It has recently been suggested that cyclooxygenase-2 (COX-2) and its product

prostaglandin E2 down-regulate the function of activated CD8+ T cells and induce their

senescence [174]. This effect may explain how the abrogation of COX-2 signalling

reduces tumour growth in mouse models of glioma [175] and mammary cancer [176]. In

light of these findings, it is tempting to study whether CD8+ T cells present in HPV-induced

lesions are affected by COX-2 inhibition.

In this study, the aim was to examine the effect of a selective COX-2 inhibitor,

celecoxib, on the infiltration and activation of CD8+ T lymphocytes in HPV-induced lesions.

For this purpose, chest skin lesions of K14-HPV16 transgenic mice were employed [73].

These mice have been designed to target the expression of HPV16 oncogenes to skin

and keratinized mucosal keratinocytes. The multi-stage disease progression in these mice

greatly resembles HPV-associated disease in cancer patients, both morphologically and

48

molecularly [76], making K14-HPV16 mice a particularly useful model for HPV research.

Material & Methods

Animals

Generation of K14/HPV16 mice on a FVB/n background has been previously

reported [73]. K14-HPV16 transgenic mice were kindly donated by Drs. Jeffrey Arbeit and

Douglas Hanahan (University of California) through the USA National Cancer Institute

Mouse Repository. The animal experiments were approved by the Universidade de Trás-

os-Montes e Alto Douro Ethics Committee (10/2013) and the Portuguese General

Veterinary Directorate (approval no. 0421/000/000/2014). Animals were maintained and

bred according to Portuguese (Decreto-Lei 113, August 7th) and European (EU Directive

2010/63/EU) legislation, under controlled conditions of temperature (23 ± 2 ºC), light-dark

cycle (12h light/12h dark) and relative humidity (50 ± 10 %), using corncob bedding. Food

and water were provided ad libitum.

Mice genotyping



assessed. HPV16-E6 and -E2 genes were amplified to confirm the presence of HPV DNA

and a fragment of mouse β-globin was also amplified as endogenous control. Lengths of

the amplicons were confirmed by agarose gel electrophoresis. Only hemizygous females

were used for the transgenic mouse groups in the experiment.

Experimental Design

Fifty-four 18 to 20 weeks-old female mice were divided into four experimental

groups, according to their genotype and taking into consideration that HPV transgenes

and celecoxib (CXB) administration could induce some mortality: group 1 (HPV16-/-

untreated animals, n = 12), group 2 (HPV16+/- untreated animals, n = 12), group 3

(HPV16-/- CXB-treated animals, n = 15) and group 4 (HPV16+/- CXB-treated animals, n =

15). All surviving mice were humanely euthanized at 24-26 weeks of age by

intraperitoneal pentobarbital overdose, followed by exsanguination by cardiac puncture.

Chest skin samples (approximately 4 cm2) were collected for flow cytometry analysis and

matched skin samples were collected for histological analysis.

49

Celecoxib administration

Celecoxib (Pfizer, New York, NY) was dissolved in drinking water at a

concentration of 0.5 mg/ml, estimating an average daily water intake of 5.0ml per mouse,

and a dose of 46.7 mg/kg/day and 2.5 mg/animal/day in an average mouse weighting 30

g. This is a well-tolerated moderate dose, as shown in previous assays [177]. However,

K14-HPV16 animals dramatically increased their water intake when CXB was added

(possibly due to the highly palatable lactose present in the vehicle) reaching up to 15 ml

per animal. The CXB concentration was thus reduced from the start of the third week

onwards down to 0.2 mg/ml, resulting in a decrease in consumption and an effective dose

of 93 mg/kg/day and 2.8 mg/animal/day. The average dose during the overall

experimental period was thus 124 mg/kg/day and 3.72 mg/animal/day.

Histological Analysis

Skin samples were fixated in 10% neutral buffered formalin for 48 hours. Samples

were dehydrated through graded alcohols and xylene and paraffin embedded in an

automatic STP 120 processor (Micron, Boise, ID). 2 µm-thick sections were stained with

haematoxylin-eosin (H&E) for histological evaluation on light microscopy. Skin samples

were classified as normal skin, epidermal hyperplasia and epidermal dysplasia.

Preparation of Single-Cell Suspensions

Chest skin samples were cut into small pieces after excessive blood vessels and

fat tissue removal. Skin fragments were incubated for 2 hours with 125 U/ml type I



buffer (all from Sigma, St. Louis, MO) and 10% foetal bovine serum (BioWest, Nuaillé,

France) at 37 ºC and 150 rpm in a 3031 orbital incubator (GFL, Burgwedel, Germany).

Subsequently, the resulting cell suspension was filtered and centrifuged at 300 � for 10

min at 4 ºC. Cells were ressuspended in phosphate buffered saline containing 1% bovine

serum albumin and 20 mM sodium azide followed by flow cytometry analysis.

Immunophenotyping

Following cell isolation, the surface phenotype of the collected cells was assessed

by flow cytometry using specific monoclonal antibodies (mAb). Cells were incubated with

anti-mouse CD16/CD32 mAb for FcγR blocking, to prevent non-specific antibody binding.

Next, cells were incubated with anti-CD8 mAb phycoerythrin-cychrome 5-conjugate (clone

53-6.7, BD Biosciences, San Diego, CA) and anti-CD107a (LAMP1) mAb phycoerythrin-

50

conjugate (clone eBio1d4b, eBioscience, San Diego, CA). Following extracellular staining,

the cells were washed, fixed in 2% formaldehyde and washed with phosphate buffered

saline containing 1% bovine serum albumin and 20 mM sodium azide. Antibody-labelled

cells were analysed in an EPICS XL flow cytometer using the EXPO32ADC software

(Beckman Coulter, Miami, FL). The assembled data files were analysed using the FlowJo

software v10.0.7 (FLOWJO, LLC, Ashland, OR).


Statistical analyses were performed using the GraphPad software (version 6.0,

GraphPad Software, Inc. La Jolla, CA). In column and dot graphs each point represents

individual mice, while bars represent the mean for the respective group. Statistical

analysis between group pairs was done using the Mann-Whitney test.

Results

General findings

All K14-HPV16 mice showed typical cutaneous changes, including diffuse

hyperkeratosis and erythema. Celecoxib-treated HPV+/- mice (group 4) showed significant

mortality: 10 out of 15 mice (66.7%) succumbed before the end of the study. All mice in

groups 1, 2 and 3 survived until the end of the study.

Histological analysis

Histological analysis of skin samples (Table 2) showed that all WT mice, groups 1

and 3, presented normal skin histology (Fig. 9a). In groups 2 and 4 (untreated and CXB-

treated HPV+/- mice, respectively), the totality of mice showed simple to papillary, diffuse,

variably severe epidermal hyperplasia extending to the follicular infundibula and

papillomatosis with orthokeratotic hyperkeratosis (Fig. 9b). Additionally, 2 animals (16.7%)

from group 2 presented multifocal epidermal dysplastic foci with parakeratotic

hyperkeratosis within the hyperplastic background (Fig. 9c). There were signs of mild

inflammation, with a small amount of macrophages, mast cells and lymphocytes in the

superficial dermis. Dysplastic foci were associated with increased numbers of leukocytes

infiltrating the dermo-epidermic junction and with intense angiogenesis.

51

Table 2: Histological classification of HPV16-induced skin l esions from 24-26 weeks-old female mice. Celecoxib –

CXB.

Group Cutaneous lesions

Incidence (%)

Normal skin Epidermal hyperplasia Epidermal dysplasia

1 (HPV-/-, n = 12) 12/12 (100%) 0/0 (0%) 0/0 (0%)

2 (HPV+/-, n = 12) 0/0 (0%) 12/12 (100%) 2/12 (16.7%)

3 (HPV-/- + CXB, n = 15) 15/15 (100%) 0/0 (0%) 0/0 (0%)

4 (HPV+/-+ CXB, n = 5) 0/0 (0%) 5/5 (100%) 0/0 (0%)

Fig. 9: Histopathological changes induced by HPV16 oncogene s in FVB/n mice, H&E. a – WT animal; Normal skin histology, 100 �. b – CXB-treated HPV16 +/- animal; Epidermal hyperplasia extending to the fol licular infundibulum and isthmus, 100 �. c – Untreated HPV16 +/- mouse; Epidermal dysplasia, 200 �. Note marked parakeratotic hyperkeratosis, loss of cell polarity, enhanced ani sokaryosis and mitotic activity. The dermal-epiderm al junction is obscured by severe inflammatory cell infiltration.

CTL infiltration and activation in celecoxib-treate d mice

With the purpose to study CD8+ T cell infiltration in HPV-induced lesions, lymphoid

cells from mice chest skin tissue were isolated and a flow cytometry analysis of the

recovered cells was performed (Fig. 10a). Skin samples from untreated HPV+/- mice

showed a significantly higher percentage of CD8+ T cells compared with untreated WT

animals (P � 0.01) (Fig. 10b). Also, although statistical significance was not achieved,

CXB-treated WT mice showed decreased CD8+ T cell infiltration when compared to CXB-

treated HPV+/- animals and the same was observed between samples from WT mice

treated with CXB (group 3) and untreated WT animals (group 1) (Fig. 10b). Moreover,

HPV+/- mice treated with CXB (group 4) showed less CD8+ T lymphocytes compared with

untreated transgenic animals (group 2) (P � 0.01) (Fig. 10b).

Next, the objective was to study whether the CD8+ T lymphocytes found in mouse

skin samples showed signs of cytotoxic activity (Fig. 10a). Thus, expression of CD107a

(LAMP1) at the cell surface was assessed by flow cytometry. CD107a reaches the surface

of CTL when lytic granules are released, exposing their membrane proteins during

52

exocytosis [144]. Thus, this lysosome-associated membrane protein is frequently used as

an immunological marker of CTL degranulation [145]. Fig. 10c shows a significantly higher

percentage of CD8+CD107a+ cells in samples from HPV+/- mice treated with CXB (group

4) when compared with untreated HPV+/- (group 2) (P � 0.01).

Fig. 10: Percentage of CD8 + and CD8+CD107a+ T cells within total lymphoid-gated cells obtained from chest skin samples from WT and K14-HPV16 transgenic mice. a – Representative analysis of the gating strategy empl oyed. Numbers within graphs correspond to the percentage of the gated population. b – Percentages of CD8 + T cells and c – percentages of CD8 +CD107a+ T cells in gated CD8 + T cells were determined by flow cytometry after sk in tissue collection and digestion with collagenase. Chest sk in samples were collected from WT and HPV +/- mice (Groups 1 and 2, respectively) and CXB-treated (CXB) WT and HPV+/- mice (Groups 3 and 4, respectively) at 24-26 weeks of age. Group 1, n = 5; Group 2, n = 5; Group 3, n = 5 ; Group 4, n = 5. Each dot represents an individual animal. Bars represent the mean value in each group. ** P � 0.01.

53

Discussion

The development of HPV-associated malignancies depends on a persistent HPV

infection. Ultimately, the ability of the immune system to eliminate the virus is the key

element to decide whether a HPV infection is cleared or evolves to cancer [2]. HPV

presents well-known mechanisms to evade host immunity and delay its elimination, thus

facilitating viral persistence [154]. When the virus is detected, an innate immune response

occurs, and leads to the development of an adaptive immune response. A fundamental

part of this adaptive response is cell-mediated immunity, characterized by the activity of a

vast number of CD8+ and CD4+ T lymphocytes [39]. In fact, infiltration by these cells in

HPV-associated lesions has already been shown to lead to regression [148, 178]. CTL

infiltration drives the response induced by an experimental therapeutic vaccine in patients

with cervical intraepithelial lesions [171] and correlates with a better prognosis in patients

with HPV-positive head and neck cancer [155, 172, 173].

Chronic inflammation is a key feature associated with carcinogenesis, namely in

the case of HPV infection [8]. COX-2 is an enzyme with an imperative role in the

metabolism of arachidonic acid, which leads to the production of prostaglandins, which in

turn promote inflammation [129]. In fact, COX-2 is over-expressed in several

malignancies, including cervical cancer [130-134]. As inflammation is known to contribute

for cancer progression [8], COX-2 inhibition is expected to result in tumour growth

inhibition whilst reducing inflammation. Some non-steroidal anti-inflammatory drugs, like

aspirin and ibuprofen, have already shown promising results as anti-tumour therapy in

both patients and pre-clinical animal models [179-182]. However, these drugs have very

low specificity. Selective COX-2 inhibitors such as CXB and rofecoxib have already been

used to prevent the development of colorectal adenomas [183, 184]. Furthermore, this

COX-2 inhibitor has been showing promising results in a tumour model of human colon

cancer when combined with chemotherapeutic drugs [185].

In this study, the effect of the selective COX-2 inhibitor CXB over the trafficking

and activation of CD8+ T lymphocytes was examined in the K14-HPV16 mouse model, a

proper model to study HPV-induced carcinogenesis due to its great similarities with the

human clinical disease [76]. High mortality was observed among CXB-treated animals,

presumably due to CXB-related toxicity.

The results presented herein show that CXB reduces the number of tumour-

infiltrating CD8+ T cells when compared with untreated mice. Still, despite the decrease in

cell numbers, CXB-treated mice have a higher percentage of activated and degranulating

CTL compared with untreated animals. These findings suggest that CXB reduces the

number of tumour-infiltrating CD8+ T cells while enhancing their effector functions. These

54

data are in agreement with a previous study using glioma-bearing mice where COX2-/-

mice had increasing percentages of tumour-infiltrating CD8+CD107a+ lymphocytes [175].

This may be explained by another study reporting that COX-2 activity leads to CD8+ T cell

senescence and this trajectory may be opposed by COX-2 inhibition, as shown by

increased levels of CD28 and interleukin-2 in CD8+ T cells [174].

Furthermore, hyperplastic epidermal lesions were evident in all surviving

transgenic mice, whilst all WT animals presented normal skin histology, as expected.

Multifocal dysplastic epidermal lesions were restricted to untreated HPV+/- animals. These

findings suggest that CXB blocked tumour progression at the hyperplastic stage, but the

small number of dysplastic lesions observed does not allow for any definitive conclusions.

In fact, COX-2 inhibition boosted the efficacy of a DNA vaccine expressing the HPV E7

oncogene, by enhancing tumour-infiltrating CD8+ T cells and slowing tumour growth [186].

However, this study was performed in mice bearing allografted TC1 lung cells

immortalized by the HPV16 E6 and E7 oncogenes and transformed by the c-Ha-ras

oncogene. Comparisons between this model and K14-HPV16 mice are limited, because

allografts do not reproduce HPV-associated multi-step carcinogenesis, being directly

implanted in the subcutis with a fully malignant phenotype. Also, keratinocytes and not

lung cells are the targets for papillomavirus infection.

It remains unclear whether CTL are activated at regional lymph nodes or at the

lesion location and this would be an interesting point to address in the future. The results

presented herein suggest that CXB induces augmented CTL degranulation in HPV-

induced lesions, possibly contributing to prevent malignant progression in this animal

model.

Conclusion

The present data confirm the potential of CXB to enhance degranulation by

tumour-infiltrating CD8+ T lymphocytes. Moreover, the effect of CXB seems to be more

complex than previously thought, as it also reduced the overall number of tumour-

infiltrating CD8+ T cells. Future studies addressing the impact of COX-2 inhibitors on the

prognosis of patients bearing HPV-induced lesions should take into account both the

number of infiltrating CD8+ T cells and their activation status.

55

Chapter 6: General Discussion and Conclusions

57

HPV-associated malignancies are a genuine health problem worldwide, with

emphasis on the increase in the number of HPV-associated oropharyngeal carcinomas

[152]. Prophylactic vaccination remains the most effective method to control HPV

infections. Currently, there are two effective commercialized vaccines: Cervarix™

(GlaxoSmithKline Biologicals, Rixensart, Belgium), a bivalent HPV16/18 vaccine; and

Gardasil™ (Merck Vaccines, West Point, PA), a quadrivalent HPV6/11/16/18 vaccine.

Both are HPV L1 virus-like particles (VLP) vaccines and they act by boosting the

production of neutralising antibodies directed against the L1 capsid protein [187]. Despite

their effectiveness, these vaccines present a few limitations, as the virus-type restriction.

The first is restricted to two HPV types (bivalent) and the second is restricted to four types

(quadrivalent). Looking to increase type-specific protection, Merck managed to create the

nonavalent HPV L1 VLP vaccine by adding the VLPs from five oncogenic HPV types (31,

33, 45, 52 and 58) in addition to the four types used in the quadrivalent vaccine. This

resulted in a 20% increase in protection, in addition to the 70% obtained from the

quadrivalent vaccine, in a total of approximately 90% protection against cervival cancer

[188]. However, despite these advances in disease prevention, an effective therapeutic

vaccine against HPV-induced malignancies remains absent.

In order to develop a therapeutic strategy, it is necessary to gather a large amount

of information on the features of disease progression as well as of the events occurring in

the lesions microenvironment. It is accepted that the carcinogenesis process induced by a

HPV infection is marked by the existence of a prominent immune response [11, 18]. Thus,

a great deal of cellular populations and chemical factors are present and play a specific

role in carcinogenesis, either promoting or impairing it. At the end, it should be the

balance between all pro- and anti-tumour stimuli that will decide whether the viral infection

is cleared or is allowed to progress towards malignancy. Therefore, a therapeutic vaccine

could possible enhance the action of anti-tumour effectors and/or inhibit the function of

known pro-tumour mediators.

Cytotoxic CD8+ T lymphocytes, for instance, are the main cells associated with the

elimination of virus-infected and transformed cells. These cells are part of the adaptive

immune response and depend on antigen presentation by MHC I molecules and other co-

stimulatory signals to become activated and exert their functions [116, 117]. However,

during the course of HPV infection, expression of MHC I molecules is usually down-

regulated. This is one of the most common immune evasion mechanisms employed by

the HPV E5 oncoprotein [154]. Infiltration of CD8+ T cells in HPV-positive lesions has

already been associated with better prognosis when compared with HPV-negative ones

[155, 172, 173, 189]. Thus, boosting the activity of CD8+ T lymphocytes seems a possible

therapeutic strategy to eliminate HPV-associated lesions. Still, the HPV immune evasion

58

mechanisms, namely MHC I molecules down-regulation, should by some means be

reversed in order to increase the efficacy of the CTL boosting.

Nevertheless, modulation of a lesion microenvironment (particularly, the CD8+ T

cells) can be accomplished by a great variety of substances such as toxins or medical

drugs. For instance, humans can become easily exposed to bracken (Pteridium spp.) –

and more importantly to its major toxin, ptaquiloside – through bovine and ovine milk and

meat consumption or ingestion of contaminated water [161-164]. Ptaquiloside can cause

several immunosuppressive events such as neutropenia, diminished NK cell cytotoxicity,

reduced expansion of antigen-specific T lymphocytes and B-cell lymphoproliferative

malignancy [125-128]. However, its effect over CTL or HPV-induced lesions was not

addressed until this moment. In turn, the effect of the selective COX-2 inhibitor celecoxib

over CD8+ T cells has already been examined in a number of studies albeit not in the

context of HPV-induced lesions and with inconsistent results [136-140]. This anti-

inflammatory drug appears as a possible therapeutic drug against inflammation-

associated cancers where COX-2 is over-expressed.

The K14-HPV16 transgenic mouse model stands out as an excellent replica to

study HPV-associated carcinogenesis. Its greatest advantage is the targeted expression

of the entire HPV early region genes to skin and keratinized mucosal keratinocytes by the

cytokeratin-14 promotor. This model expresses the virus proteins in the same cell types

that are infected by HPV in human patients, therefore supporting the use of this model.

Furthermore, the microscopic morphologic similarities between this mouse and the human

disease also validate its appliance in HPV-induced carcinogenesis research [76].

Therefore, the K14-HPV16 mice can be employed in the study of the immune response

against HPV and, more precisely, in the disclosure of the infiltrating cells in the lesions

microenvironment. In addition, this model is appropriate to study the immunomodulatory

effects of multiple substances at the lesions microenvironment. Hence, in this work, these

transgenic mice were utilized to analyse the infiltration and activity of CD8+ T cells in HPV-

induced lesions at different ages and after the treatment with two distinct

immunomodulatory agents.

The results presented herein suggest that CD8+ T lymphocytes heavily infiltrate

HPV-induced lesions. These cells show signs of cytotoxic activity and the number of

degranulating CTL increases in response to more aggressive lesions. However, CTL

infiltration and degranulation were not sufficient to induce lesion regression. This may be

related to the model now employed, in which all keratinocytes express the HPV

oncogenes. In these conditions, even if many neoplastic keratinocytes are cleared by the

immune response, there are no normal cells to replace them, but only an endless

population of transformed cells that will sustain tumour progression.

59

Furthermore, the CD8+ T cell response can be modulated. Ptaquiloside impaired

the cytotoxic activity of these cells and diminished the differentiation of memory CTL.

Apparently, ptaquiloside promoted the development of more aggressive lesions, although

this correlation could not be statistically confirmed. This is likely to constitute another

mechanism through which ptaquiloside contributes to the persistence and the malignant

transformation of viral lesions.

In contrary, celecoxib significantly enhanced the release of lytic granules by CTL,

even though greatly reducing the number of infiltrating lymphocytes. However, increased

cytotoxicity was not perceptibly associated with lesion regression, which may be due to

the model limitations, as outlined above. Nonetheless, the focus of this work was the

characterization of the CD8+ T cell infiltrate. Thus, additional studies are required to

understand why CTL activity is not sufficient to induce the regression of HPV-associated

lesions.

In the near future it will be worth exploring which other immune cellular populations

may also infiltrate HPV-induced lesions and what soluble mediators they could be

producing. This knowledge should provide a better characterization on the pro- and anti-

tumour stimuli in the lesion microenvironment, therefore helping to decide which cells

should be boosted and which should be inhibited in order to attain regression. In

conclusion, this work once more recognizes the value of the K14-HPV16 transgenic

mouse model for HPV-induced carcinogenesis research and it should also be a very good

model for the testing of new therapeutic strategies. Finally, the results presented here are

encouraging in regard to the search for lesion regression, as they show high infiltration

and activity of the most common anti-tumour immune effector cells.

61

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