Modulating The CD8+ T Cell Response in Human Papillomavirus-Induced Lesions: Studies in The K14-HPV16 Transgenic Mouse Model

Disse ação de candida u a ao g au de Mes e em Oncologia – especialização em Oncologia Molecula – subme ida ao Ins i u o de Ciências Biomédicas de Abel Salaza da Uni e sidade do Po o. O ien ado – P o esso Dou o Rui Gil da Cos a Ca ego ia – In es igado A iliação – LEPABE, Faculdade de Engenha ia da Uni e sidade do Po o; G upo de Oncologia Molecula e Pa ologia Vi al, CI-IPOP, Ins i u o Po uguês de Oncologia do Po o Co-O ien ado – P o esso Dou o Rui Medei os Ca ego ia – P o esso Con idado Associado A iliação – Ins i u o de Ciências Biomédicas de Abel Salaza ; G upo de Oncologia Molecula e Pa ologia Vi al, CI-IPOP, Ins i u o Po uguês de Oncologia do Po o Co-O ien ado – P o esso Dou o Manuel Vilano a Ca ego ia – P o esso Associado A iliação – Ins i u o de Ciências Biomédicas de Abel Salaza CARLOS EDUARDO REIS DOS SANTOS MODULATING THE CD8 + T CELL RESPONSE IN HUMAN PAPILLOMAVIRUS-INDUCED LESIONS: STUDIES IN THE K14- HPV16 TRANSGENIC MOUSE MODEL II III Alguns dos esul ados incluídos nes a disse ação de mes ado o am subme idos pa a publicação: - San os C., Fe ei inha P., Sousa H., Ribei o J., Bas os M.M.S.M., Faus ino-Rocha A.I., Oli ei a P.A., Medei os R., Vilano a M., Gil da Cos a R.M. (2015) Kine ics o CD8 + T cells in human papilloma i us-induced lesions – da a om K14-HPV16 ansgenic mice. Submi ed o he jou nal Tumo Biology wi h he numbe TUBI-D-15-04094. - San os C., Ne o T., Fe e inha P., Sousa H., Ribei o J., Bas os M.M.S.M., Oli ei a P.A., Medei os R., Vilano a M., Gil da Cos a R.M. (2015) Celecoxib p omo es deg anula ion o CD8 + T cells in HPV-induced lesions o ansgenic mice. Submi ed o he jou nal An i i al Resea ch wi h he numbe AVR-D-16-00002. Alguns esul ados o am ambém publicados em a as de um cong esso in e nacional: - San os C., Fe ei inha P., Sousa H., Ribei o J., Bas os M.M.S.M., Faus ino-Rocha A.I., Oli ei a P.A., Medei os R., Vilano a M., Gil da Cos a R.M. (2015) Role o CD8 + T cells in HPV-induced skin cance : da a om a K14-HPV16 ansgenic mouse model; P oceedings o he HPV2015 – 30 h In e na ional Papilloma i us Con e ence & Clinical and Public Heal h Wo kshops; 17 h -21 s Sep embe 2015, Lisbon, Po ugal. (Bes pos e p esen a ion awa d) IV V “When you wan o succeed as bad as you wan o b ea he hen you’ll be success ul” – E ic Thomas VI VII Ag adecimen os Nes e momen o, que ma ca o inal de mais uma e apa da minha ida, não pode ia deixa de ag edece às pessoas que de alguma o ma con ibuí am pa a a conclusão des a e apa. Es e úl imo ano oi bas an e desa ian e e exigen e e odo o conhecimen o e apoio que ecebi oi undamen al pa a chega a é aqui. Em p imei o luga , gos a ia de ag adece à pessoa que me ecebeu e que me acompanhou mais de pe o no desen ol imen o des e abalho, o meu o ien ado , P o esso Dou o Rui Gil da Cos a. Ag adeço a sua con iança em mim pa a le a a cabo es e abalho, odos os conhecimen os que me ansmi iu, a disponibilidade pa a ajuda no que osse p eciso, odos os conselhos e a cons an e boa disposição. Ao P o esso Dou o Rui Medei os, meu co-o ien ado , ag adeço oda a ajuda o necida ao longo des e ano e po udo o que me ensinou. Mesmo es ando cheio de abalho a anja a manei a de me ecebe semp e que apa ecia no seu gabine e do IPO pa a me pode ajuda no que eu p ecisasse e semp e bem-dispos o. Ao P o esso Dou o Manuel Vilano a, ambém meu co-o ien ado , po udo aquilo que con inua a ensina -me, pelo seu igo que an o ap ecio, pela disponibilidade que a anja semp e que eu p eciso de ala consigo mesmo quando a quan idade de abalho não o “deixa espi a ”. A es e conjun o de o ien ado es ag adeço odas as discussões das quais su gi am ideias de abalho, ag adeço udo o que me ensina am e, acima de udo, ag adeço a comp eensão e o apoio que semp e demons a am pe an e a minha ida de es udan e- a le a. Não só comp eendem como ambém alo izam o que aço e é ealmen e bom sen i esse apoio. Ao Ped o Fe ei inha, mais do que um co-o ien ado no passado, um amigo e colega in es igado no p esen e. Mui o do que ap endi sob e abalha num labo a ó io de o-o a ele. Pa a além de udo o que me ensinou e con inua a ensina es á semp e dispos o a ajuda no que o p eciso. Não só é dedicado ao abalho como ambém é uma pessoa óp ima pa a i oma um ca é e e uma con e sa animada. Às es an es pessoas que abalham no Labo a ó io de Imunologia Má io A ala Cha es (ICBAS), em especial à Enca nação Rebelo, Joana Al es e Alexand a Co eia, ag adeço o ca inho e a boa disposição de odos os dias, a disponibilidade pa a ajuda no que p ecisasse e as con e sas aleg es e en iquecedo as. À P o esso a Paula Oli ei a, à Ana Faus ino-Rocha e ao Tiago Ne o ( odos da Uni e sidade de T ás-os-Mon es e Al o Dou o) ag adeço odo o apoio que me de am du an e a ealização dos abalhos expe imen ais. VIII Po úl imo, mas mui o impo an es, a amília e os amigos o mam um conjun o de pila es que se em de apoio nos momen os mais complicados e de di e são quando a ocasião o pe mi e. Àqueles que me acompanha am mais de pe o du an e es e ano mesmo, em alguns casos, es ando longe (Tiago Sil a, Bea iz Sil a, Helena Pinhei o, Ma iana Pe ei a, Inês Sousa, Hildebe o Mo ei a, Ana Rosa, Luciana Lei e, Ri a Fa ia, Miguel Gonçal es, Cá ia Oli ei a, Joana Pin o, Rena a Lopes, Diogo Edi, Isabel Pai a, Ca a ina Pe ei a e, cla o, aos meus colegas de equipa) ag adeço pela o ça que me de am quando as coisas pa eciam não co e bem, a mo i ação que me passa am quando es a me al a a, as con e sas en iquecedo as sob e o abalho, os momen os de descon ação, ga galhada e di e são quando es es e am possí eis e, p incipalmen e, po se em e dadei os amigos e comp eende em os sac i ícios que aço em p ol dos meus objec i os e ambições enquan o es udan e-a le a. Aos meus pais e i mã, ag adeço po udo aquilo que não é possí el ag adece o su icien e. Ag adeço pela educação que me de am e po udo o que me ensina am, po odas as condições que semp e me de am pa a o meu c escimen o, pelas chamadas de a enção, po exigi em o melho de mim, po me apoia em em odas as decisões, po me o ien a em. Se hoje posso dize que enho o gulho no que me o nei de o-o in ei amen e a eles. Ob igado. IX Index Ag adecimen os .............................................................................................................. VII Index ................................................................................................................................ IX Abb e ia ions ................................................................................................................... XI Resumo ......................................................................................................................... XIII Abs ac .......................................................................................................................... XV Chap e 1: In oduc ion ..................................................................................................... 1 The Basis o HPV In ec ion and Cell T ans o ma ion ..................................................... 4 HPV In ec ion s. Hos Immune Response .................................................................... 5 HPV-associa ed Ch onic In lamma ion .......................................................................... 6 HPV-induced Ca cinogenesis: The Bes Models o Unde s and I ................................. 9 K14-HPV16 T ansgenic Mice as a Model o In lamma ion-associa ed Ca cinogenesis12 Modula o s o he immune esponse and hei e ec o e CD8 + T cells ........................15 Chap e 2: Objec i es ......................................................................................................17 Chap e 3: Kine ics o CD8 + T cells in human papilloma i us-induced lesions..................19 In oduc ion ..................................................................................................................21 Ma e ial & Me hods ......................................................................................................22 Animals .....................................................................................................................22 Mice geno yping........................................................................................................22 S udy Design ............................................................................................................22 His ology ...................................................................................................................22 P epa a ion o Single-Cell Suspensions ....................................................................23 Immunopheno yping .................................................................................................23 S a is ical Analysis ....................................................................................................23 Resul s .........................................................................................................................24 T ansgenic Mice Show Epide mal Hype plasia and Dysplasia ..................................24 Inc eased CD8 + T lymphocy es numbe s and ac i a ion in HPV16 +/- mice ................24 Discussion ....................................................................................................................26 Conclusion ...................................................................................................................27 Chap e 4: P aquiloside inhibi s umou -in il a ing CD8 + T cells in HPV- ansgenic mice .29 In oduc ion ..................................................................................................................31 Ma e ial & Me hods ......................................................................................................32 Mice ..........................................................................................................................32 XVI 1 Chap e 1: In oduc ion 2 3 Human papilloma i us (HPV) is he pa hogen esponsible o he mos common sexually ansmi ed in ec ion among bo h men and women in he Uni ed S a es [1]. Mos sexually ac i e people will be in ec ed by HPV du ing hei li e. The as majo i y o HPV in ec ions in immunocompe en indi iduals a e clea ed by he immune sys em, bu in app oxima ely 10% o cases [2, 3] he pe sis ence o his in ec ion will cause he de elopmen o disease and p og ession o cance . HPV has opism o skin and mucosal epi helia especially om he anogeni al and o opha yngeal a eas. High- isk HPVs (mos ly ypes 16, 18, 31 and 33) ac as c ucial e iologic agen s in ce ical ca cinoma [4] – which is he six h mos equen malignancy in women wo ldwide [5] – and a e he cause o some head and neck neoplasms whe e incidence depends on he coun y and o he associa ed isk ac o s like obacco [6, 7]. Since 1863 [8], when Vi chow i s hypo hesised a link be ween in lamma ion and cance , many s udies ha e been ca ied ou in o de o unde s and he unde lying mechanisms o his ela ion and i s in e enien s. The impo ance o his ela ion was u he ein o ced by he awa d o he Nobel P ize o Robin Wa en and Ba y Ma shall o hei wo k in disco e ing Helicobac e pylo i as he e iologic agen o in lamma ion- associa ed gas ic cance [9]. The umou mic oen i onmen is cha ac e ised by he p esence o a a ie y o leucocy es which ha e been shown o be a ac ed by chemokines p oduced by umou and s omal cells [10]. Lymphocy es a e an impo an pa o in lamma o y in il a es in he umou mic oen i onmen [8, 11] and di e en lymphocy ic popula ions can play di e en oles. Fo ins ance, T helpe (Th) 1, Tγδ and cy o oxic T cells a e associa ed wi h disease- ee su i al. In con a y, Th2, Th17 and T egula o y (T eg) cells a e allied wi h a poo e ou come [12]. Neu ophils, umou - associa ed mac ophages (TAMs), dend i ic cells (DCs) and mas cells ep esen o he leucocy e popula ions also equen ly ound in he umou en i onmen ha may play dual oles du ing ca cinogenesis [8, 13]. These umou -associa ed in lamma o y cells, oge he wi h he umou cells, p oduce and sec e e a wide se o cy okines (e.g.: umou nec osis ac o (TNF)-α, in e leukin (IL)-1, IL-6 and monocy e colony-s imula ing ac o ) and chemokines (e.g.: monocy e chemo ac ic p o ein-1) ha may ei he help umou p og ession o con ain i [14-17]. In he case o HPV-associa ed ca cinogenesis, he i us in ec s ke a inocy es and an immunological esponse a ises as cy okines (mos ly TNF-α, IL-1 and ype I and II in e e ons – IFNs) a e eleased by he in ec ed ke a inocy es and local immune cells [18]. Howe e , HPVs may a oid he e ec o unc ions o p o-in lamma o y cy okines by di e en mechanisms [19-22]. Thus, i he in ec ion pe sis s, he chance o neoplas ic ans o ma ion ises [23]. Besides i s impo ance in he ea ly s ages o ca cinogenesis, 4 inna e and adap i e immune esponses a e c i ical ac o s in ol ed in malignan p og ession. The Basis o HPV In ec ion and Cell T ans o ma ion Human papilloma i uses ha e hei genome di ided in h ee main egions: he non-coding uppe egula o y egion, he ea ly egion encoding six known oncop o eins and he la e egion, which encodes he capsid p o eins. The oncop o eins encoded in he ea ly egion, mainly E6 and E7 bu also E5, a e key pa icipan s in he in ec ious cycle and a e he sou ce o he gene ic al e a ions which po en ially lead o HPV-associa ed ca cinogenesis as e iewed elsewhe e [24]. The E5 oncogene is belie ed o ha e low impac in he main enance o he malignan pheno ype o ce ical cance cells as i appea s o be dele ed in a ious ca cinomas [25]. Ne e heless, when p esen , i akes an impo an pa in mul iple mechanisms a ou ing neoplas ic ans o ma ion. Some in i o s udies ha e shown ha he HPV16 E5 oncop o ein can p omo e cell cycle p og ession and DNA syn hesis by down- egula ing he exp ession o umou supp esso p o eins p21 and p27 [26, 27]. Mo eo e , his oncop o ein is cen al o e asion o he hos immunosu eillance. In E5-exp essing cell lines an igen-p esen ing majo his ocompa ibili y complex (MHC) class I molecules appea o be apped in he Golgi appa a us which educed hei su ace exp ession [28, 29]. The down- egula ion o MHC class I on cells exp essing E5 ul ima ely impai s hei ecogni ion and clea ance by cy o oxic T lymphocy es (CTL). The ea ly genes E6 and E7 a e known o ha e g ea oncogenic po en ial. They play a pi o al ole in HPV-media ed ca cinogenesis as hey eac wi h impo an umou supp esso p o eins impai ing hei unc ions. Thus, E6 binds o and p omo es he deg ada ion o he p53 umou supp esso p o ein [30, 31], impai ing impo an mechanisms o de ence such as DNA epai and apop osis. Addi ionally, he E6 p o ein acili a es ca cinogenesis by educing cellula senescence (as a esul o elome ase ac i a ion [32]), ac i a ing he NF-κB pa hway and e ading he in e e on esponse [24]. The E7 oncop o ein binds o umou supp esso p o eins o he e inoblas oma amily o p o eins (pRb) and induces hei inac i a ion [33, 34], he e o e inc easing DNA syn hesis and boos ing cellula p oli e a ion. Fu he mo e, E7 is able o p omo e genomic ins abili y, which inc eases he likelihood o malignan p og ession [24]. Toge he , he E6 and E7 oncogenes a e capable o ans o ming and induce he immo aliza ion o ke a inocy es [35]. Un il oday, he h ee ea ly p o eins men ioned abo e appea o be, o all HPV p o eins, he ones wi h highe oncogenic po en ial. Gi en hei oles in e ading hos immuni y, i is impo an o unde s and how he human immune sys em esponds o HPV in ec ion. 5 HPV In ec ion s. Hos Immune Response Along he yea s, mammals ha e g own complex inna e and adap i e immune mechanisms o con ol au oimmuni y and o p o ec he hos agains mic obial in ec ions. Vi uses can be de ec ed by cellula ecep o s gene ally designa ed as pa e n ecogni ion ecep o s (PRRs), which igge inna e immune mechanisms ha subsequen ly may be ollowed by a mo e speci ic adap i e immune esponse. These cellula ecep o s a e p o icien a ecognising se e al pa hogen-associa ed molecula pa e ns, like i al DNA o RNA o bac e ial cell wall o in acellula componen s, and also in e ac ing wi h dange - associa ed molecula pa e ns like in acellula molecules eleased om damaged cells and cells unde going unp og ammed cell dea h [36]. HPVs a e double-s anded DNA i uses ha a e mos likely o be ecognized by Toll-like ecep o 9 (TLR-9). This ecep o is in acellula ly exp essed in he memb ane o endolysosomes and ecognizes unme hyla ed CpG-DNA sequences ha bou ed by pa hogens like i uses, bac e ia and p o ozoa [36]. Howe e , HPVs appea o somehow su pass hese PRRs. The oncop o eins E6 and E7 may p omo e he down- egula ion o TLR-9 as has been shown in i o o HPV16 [37]. The e o e, a he s a , he HPV in ec ion seems o occu wi hou he awa eness o he hos immune sys em [38]. Fu he mo e, in case o a pe sis en in ec ion, he i us impai s immune cell unc ions as well as se e al molecula pa hways in ol ed in he immune esponse [39]. Also, o he inna e immune mechanisms, like he IFN esponse, can be ab oga ed by he ac ions o some HPV oncop o eins, as men ioned abo e. HPV is ansmi ed h ough skin- o-skin con ac , usually h ough mic o wounds. The e exis s a a ie y o immune cells di ided be ween he de mis and epide mis. In he epide mis, he main cellula popula ion a e ke a inocy es bu he e can also be ound Lange hans cells and T lymphocy es [39]. Ke a inocy es, he main a ge s o HPV, a e capable o some immune unc ions. These cells can p oduce and elease cy okines, induce he ac i a ion o memo y T cells and may also ac as non-p o essional an igen- p esen ing cells (APCs) [40]. Hence, ke a inocy es a e o g ea impo ance in he ini ia ion o an immune esponse agains HPV in ec ions. On he o he hand, he majo i y o he so- called “classical” immune cells (mac ophages, DCs, T lymphocy es and o he s) can be ound in he de mis [39]. Bo h ke a inocy es and immune cells on si e a e capable o p o- in lamma o y cy okine p oduc ion [41] and an igen p esen a ion in esponse o se e al pa hogens. Al hough he inna e immune esponse o HPV in ec ions emains poo ly unde s ood, he immunological p ocesses desc ibed abo e a e hough o be he main ones esponsible o he ini ia ion o an adap i e immune esponse, which will de e mine he clea ance o pe sis ence o he in ec ion. In ac , only abou 10% o HPV in ec ions will 6 de elop clinical symp oms and possibly e ol e o cance [2, 3]. The e o e, wa s and low g ade ce ical in aepi helial neoplasms (CIN1) a e in mos cases o ced o eg ess by he cell-media ed adap i e immune esponse [42]. I has been shown ha CD4 + and CD8 + HPV16-speci ic T cells mig a e o he skin a e in ade mal challenge wi h HPV16 pep ides [43]. This cellula mig a ion e en appea s o be ollowed by an an ibody- media ed esponse, as obse ed in HPV-in ec ed women [44]. S ill, an ibody i es a e low and many women may e en no p oduce hem. HPV-associa ed Ch onic In lamma ion The abo e desc ibed mechanisms a e hough o explain how a human immune sys em eac s since he beginning o a HPV in ec ion un il he momen o immunological clea ance. Howe e , as seen ea lie , HPV oncop o eins ha e ways o e ading he hos immune esponse. This e asion a ou s he pe sis ence o he in ec ion, which in u n may p omo e umou igenesis. In his con ex , he cellula con en o he in lamma o y in il a e is decisi e o ei he neoplas ic ans o ma ion o elimina ion o in ec ed/ ans o med cells (Fig. 1). While some cellula popula ions suppo HPV’s immune e asion and cell ans o ma ion mechanisms, o he s ha e a ole in inhibi ing malignan p og ession o e en in po en ia ing umou eg ession. Fo ins ance, T egula o y (T eg) cells acili a e he i al immune e asion. These cells display he CD4 + CD25 + FoxP3 + pheno ype and play an immunosupp essi e ole impo an o p e en sel -agg ession by he immune sys em [45]. Because o hei immunosupp essi e unc ion, T eg cells a e o en associa ed o pe sis en in ec ion and umou p og ession. Indeed, s udies ha e shown a co ela ion be ween inc eased equencies o FoxP3 + T cells a bo h sys emic [46] and ce ical [47] le el and HPV pe sis en in ec ion. Fu he mo e, a s udy has shown he chemokine CXCL12 (which is no exp essed in no mal skin) o ac as a chemoa ac an o T eg cells [48]. This wo k shows e idence ha HPV s imula es an inc eased p oduc ion o CXCL12 which d aws a ising numbe o FoxP3 + cells. Besides T eg cells, ano he subse o CD4 + T cells can ha e p o- umou unc ions. Type-2 helpe T (Th2) cells can also con ibu e o impai he p o ec i e immune esponse, p edominan ly o he Th1 ype, in he umou mic oen i onmen as hey p oduce IL-4, -5 and -13, as well as IL-10. As seen ea lie , his ype o immune esponse acili a es malignan p og ession [12]. Fu he mo e, mac ophages a e hough o be a majo componen o umou in il a es [8] and a e highly equen in HPV-associa ed umou s [49]. In he p esence o Th2- ype cy okines like IL-4 and IL-13, umou -associa ed mac ophages (TAMs) a e al e na i ely ac i a ed and acqui e he M2-pheno ype [50]. Unlike he M1 mac ophages, hese M2 cells exp ess 7 a ious ea u es and unc ions ha a e commonly associa ed wi h immune egula ion and umou p og ession. Fo ins ance, M2 TAMs, oge he wi h mas cells, a e an impo an sou ce o g ow h ac o s (e.g.: ascula endo helial g ow h ac o – VEGF), cy okines (e.g.: TNF-α, ans o ming g ow h ac o -β), p o eases (e.g.: ma ix me allop o einases) and o he molecules which in u n p omo e angiogenesis and issue emodelling [51, 52]. These e en s acili a e he in ake o nu ien s and p o ide o he s imuli which help umou g ow h, in asion and me as asis. On he o he hand, TAMs can be classically ac i a ed upon mic obial s imuli o induced by p o-in lamma o y cy okines, such as IFN-γ, o play impo an an i- umou unc ions [50]. M1- ype TAMs can, he e o e, ac as induce and e ec o cells in Th1- ype immune esponses agains umou s. They usually display an IL- 12 high , IL-23 high pheno ype and p oduce high le els o p o-in lamma o y cy okines (IL-1β, IL-6 and TNF-α) and eac i e ni ogen species [53]. Besides M1 TAMs, o he cellula popula ions play impo an an i- umou oles among he la gely he e ogeneous umou in lamma o y in il a es. T lymphocy es a e also impo an immune e ec o s. Type-1 esponses, media ed by Th1 cells ep esen he p o o ypic esponse adequa e o e adica e cance cells. The di e en ia ion o his ype o CD4 + T cells is d i en by IL-12 and hey a e cha ac e ised by he p oduc ion o p o-in lamma o y cy okines like IFN-γ. Th1 cance cells can di ec ly induce he killing o umou cells h ough TNF- ela ed apop osis- inducing ligand (TRAIL) and/o Fas Ligand (FasL) pa hways [54] and can induce he cy o oxic ac i i y o CD8 + T lymphocy es and o he e ec o cells h ough he p oduc ion o speci ic cy okines [55]. In u n, CD8 + T cells, which may di e en ia e in o cy o oxic T lymphocy es (CTL), a e commonly p o ec i e agains in acellula pa hogens, like i uses, and umou cells. These lymphocy es we e obse ed in highe equency in HPV-posi i e ca cinomas when compa ed wi h HPV-nega i e lesions [56]. Fu he , his di e ence co ela es wi h he pa ien s’ p ognosis as a highe CD8 + T cell equency is associa ed wi h augmen ed o e all su i al. Mo eo e , “na u al kille ” (NK) cells and NKT cells ac simila ly o CTL bu a e pa o he inna e immune esponse. Unlike CTL, NK cells do no equi e he p esence o MHC I molecules on a ge cells o exe hei cy o oxic unc ion. Thus, i HPV success ully down- egula es MHC I exp ession, he eby a oiding he ac ion o CTL, in ec ed cells may s ill be a ge ed by NK cells. Like CTL, NK and NKT cells exp ess IFN-γ, pe o in and g anzyme and a e hus capable e ec o s in he an i- i al immune esponse [39] as well as being able o also pa icipa e in he killing o cance cells [57]. Addi ionally, se e al APCs (like DCs and Lange hans cells) a e o g ea alue and a e equen in he umou mic oen i onmen . They a e essen ial o he ac i a ion o e ec o cells h ough an igen p esen a ion and cy okine p oduc ion [13]. 8 Fig. 1 : Schema ic ep esen a ion o he immune cell in il a e in HPV- induced ca cinogenesis. F om op o bo om, HPV in ec ion induces neoplas ic ans o ma ion wi h associa ed issue emodelling e en s which lead o hos immune esponse. This esponse could ei he con ibu e o disease p og ession o eg ession which is e lec ed in he cellula popula ions ec ui ed in ei he case. CD8 + , Th1- ype CD4 + T, NK cells and M1 mac ophages a e commonly associa ed wi h umou eg ession while, T eg, Th2- ype CD4 + T cells and M2 mac ophages a e usually ela ed o malignan p og ession. 9 The c oss- alk o immune cells wi h HPV-in ec ed cells is s ill no ully unde s ood. Howe e , i is gene ally ag eed ha he in lamma ion associa ed wi h he i al in ec ion acili a es he de elopmen o HPV-associa ed cance s. In gene al e ms, egula o y T cells, M2 TAMs, mas cells and Th2 cells mos likely ep esen he immune cells wi h he g ea es con ibu ion o pe sis en in ec ions and malignan p og ession. Addi ionally, HPV- ans o med ke a inocy es we e shown o p oduce he immunosupp essi e cy okine IL-10 in HPV-associa ed ce ical cance [58], which may be igge ed by he i us oncop o eins as an immune e asion mechanism. On he o he hand, M1 mac ophages, NK and NKT cells, CTL and Th1 cells a e, wi h he suppo o APCs, he majo pa icipan s in an i- i al esponses and umou eg ession. S ill, i is impo an o emphasize ha his whole in e ac ion in he umou milieu is g ea ly complex due o he he e ogenei y o cellula popula ions he ein, which likely jus i ies he di icul y in cha ac e ising he ela ionship be ween in lamma o y cells and HPV in ec ion. HPV-induced Ca cinogenesis: The Bes Models o Unde s and I Along he yea s, scien is s ha e been imp o ing ou unde s anding o ch onic HPV in ec ion and i s associa ion wi h ca cinogenesis. In i o and in i o animal models a e key elemen s owa ds he e olu ion o scien i ic knowledge (Table 1) and he in i o ones a e al eady widely used in HPV-induced ca cinogenesis esea ch. HPV- ela ed in i o s udies include, in mos cases, he use o single-laye cul u es o common ce ical ca cinoma cell lines (HeLa, SiHa, CaSki and o he s). In he las wo decades, a mo e sophis ica ed ype o in i o assay was de eloped and used as an al e na i e model. O gano ypic (o a ) cul u es allow he p oli e a ion and di e en ia ion o epi helial cells a an ai -liquid in e ace on a de mal-equi alen suppo [59-61]. Thus, hese a cul u es a e use ul o s udy he e en s occu ing in human s a i ied epi helia in he cou se o HPV in ec ion and HPV-induced ca cinogenesis. Alongside wi h he cell cul u e echnique, in i o animal models a e ex ensi ely exploi ed o scien i ic pu poses. On one hand, bo ine ca le, dogs and abbi s can be used o s udy bo ine, canine o al and co on ail abbi papilloma i uses, espec i ely [62, 63]. The s udy o hese animal papilloma i uses is impo an as hey a e e iologic agen s o diseases in a m and companion animals, p o ide in i o models o HPV esea ch and help o disco e new subjec s o s udy on HPV. Howe e , hese la ge animals a e no common objec s o s udy because hey a e associa ed wi h a g ea deal o inancial cos s and e hical issues. In u n, he mouse is he mos commonly used es subjec in heal h- associa ed s udies. Mice display a g ea simila i y o humans in e ms o ana omy, physiology, including he immune one, and gene ics which, in addi ion o i s cos - 16 [123]. B acken oxins a e hypo hesized o d i e he malignan p og ession o papilloma i us-induced uppe diges i e lesions in ca le and human popula ions, as ema ked by Chang e al. [124]. P aquiloside is a chemically uns able compound ha has been shown o induce splenic whi e pulp a ophy, neu openia, educed NK cells ac i i y and a B-cell lymphop oli e a i e malignancy [125-128]. Howe e , un il now, he e a e no epo s o an associa ion be ween p aquiloside and CD8 + T lymphocy es. In u n, celecoxib (CXB) is an immunomodula o y d ug which speci ically inhibi s cyclooxygenase- 2 (COX-2). This enzyme plays a key ole in he de elopmen o an in lamma o y esponse [129] and is o e -exp essed in se e al ypes o cance [130-134]. By selec i ely inhibi ing COX-2, celecoxib diminishes he p oduc ion o p os aglandins wi hou signi ican ly impai ing he COX-1 iso o m [135]. Ac ually, a ew s udies ha e al eady enligh ened he e ec o his selec i e COX-2 inhibi o o e cy o oxic T cells [136-140]. Howe e , whe he celecoxib enhances o dec eases CD8 + T lymphocy es in il a ion and ac i i y a ies be ween s udies. S ill, none o hese s udies analysed he e ec o celecoxib o e CTL in HPV-associa ed lesions. 17 Chap e 2: Objec i es The pu pose o his wo k was o assess he in il a ion and ac i i y o cy o oxic CD8 + T lymphocy es in HPV-induced skin lesions, using he K14-HPV16 mouse model. Thus, he wo k was ocused in some mo e speci ic aims: - To pe o m a kine ic s udy o he CTL in il a ion and ac i a ion by compa ing lesions om di e en -aged mice; - To analyse he immunomodula o y e ec o p aquiloside o e CD8 + T cell in il a ion and ac i a ion; - To analyse he immunomodula o y e ec o celecoxib o e CD8 + T cell in il a ion and ac i a ion. 18 19 Chap e 3: Kine ics o CD8 + T cells in human papilloma i us-induced lesions 1 1 The con en s o his chap e we e adap ed om: - San os C., Fe ei inha P., Sousa H., Ribei o J., Bas os M.M.S.M., Faus ino-Rocha A.I., Oli ei a P.A., Medei os R., Vilano a M., Gil da Cos a R.M. (2015) Kine ics o CD8 + T cells in human papilloma i us-induced lesions – da a om K14-HPV16 ansgenic mice. Submi ed o he jou nal Tumo Biology wi h he numbe TUBI-D-15-04094. 20 21 In oduc ion Human papilloma i us (HPV) is he main e iologic agen o ce ical cance [4], bu can also o igina e anal, skin and head and neck malignancies [6, 141, 142]. Only a pe sis en in ec ion by an oncogenic HPV ype (mainly HPV 16 and 18) can induce malignan ans o ma ion. In app oxima ely 90% o HPV in ec ed indi iduals he hos immune sys em is able o igh he i us and hinde i s dissemina ion. Howe e , in he emaining 10%, HPV can e ec i ely e ade immune de enses and lead o clinical disease [2, 3]. K14-HPV16 ansgenic mice, c ea ed nea ly wo decades ago [73], a e an use ul in i o animal model o he s udy o HPV-induced ca cinogenesis. This model sha es a numbe o mo phologic and molecula simila i ies o HPV- ela ed human disease [76], hus unc ioning as an excellen eplica o he mul i-s age p ocess o ca cinogenesis. Ta ge ing o HPV16 oncogenes o ke a inocy es by he ke a in-14 (K14) p omo e /enhance is he key cha ac e is ic o his model [73]. HPV-induced ca cinogenesis is associa ed wi h p og essi ely in ense ch onic in lamma ion. The e o e, a g ea di e si y o immune cells and a mul iplici y o soluble media o s can be ound wi hin he umou mic oen i onmen [18]. The in lamma o y in il a es p o ide p o- and an i- umou s imuli, which will a ou ei he he de elopmen o he eg ession o he lesion [55, 143]. CD8 + T cells a e es ic ed o majo his ocompa ibili y complex class I (MHC I) molecules which can p esen pep ides gene a ed om in acellula i uses and/o umou cells [117]. Upon ecognizing speci ic pep ides p esen ed on MHC I molecules on he su ace o p o essional an igen p esen ing cells, which also display co-s imula o y molecules, hese T cells can di e en ia e in o cy o oxic T lymphocy es (CTL), helped by cy okine s imuli [116]. CTL a e an impo an pa o umou -speci ic immuni y. They may elimina e a ge cells ei he di ec ly, h ough he elease o ly ic g anules con aining se e al enzymes (such as pe o in and g anzyme) o by he engagemen o dea h ecep o s (e.g.: FAS-FASL), and indi ec ly, ollowing an in e e on-γ-dependen mechanism which leads o cell cycle inhibi ion, apop osis and s imula ion o mac ophage an i- umou ac i i y [118, 119]. Thus, using he K14-HPV16 ansgenic mouse model, he aim was o examine he kine ics o CD8 + T cell in il a ion in HPV-induced lesions du ing mul i-s ep ca cinogenesis a di e en ime poin s. The goal was also poin ed o unde s and whe he hese cells we e ac i a ed by de e mining he p esence o he lysosome-associa ed memb ane p o ein 1 (LAMP1), also known as CD107a, a he cell su ace. 22 Ma e ial & Me hods Animals Gene a ion o K14-HPV16 mice on a FVB/n backg ound has been p e iously epo ed [73]. K14-HPV16 ansgenic mice we e gene ously dona ed by D s. Je ey A bei and Douglas Hanahan (Uni e si y o Cali o nia) h ough he USA Na ional Cance Ins i u e Mouse Reposi o y. The animal expe imen s we e app o ed by he Uni e sidade de T ás- os-Mon es e Al o Dou o e hics commi ee (10/2013) and he Po uguese Ve e ina y Di ec o a e (0421/000/000/2014). Animals we e main ained and b ed acco ding o Po uguese (Dec e o-Lei 113/2013, Augus 7 h ) and Eu opean (EU Di ec i e 2010/63/EU) legisla ion, unde con olled condi ions o empe a u e (23 ± 2 ºC), ligh -da k cycle (12h ligh /12h da k) and ela i e humidi y (50 ± 10 %), using ha dwood bedding. Food and wa e we e p o ided ad libi um. Mice geno yping Animals we e geno yped a weaning, using ail ip samples as desc ibed p e iously [113, 114]. B ie ly, nucleic acids we e ex ac ed and DNA quali y and pu i y we e assessed. HPV16-E6 and -E2 genes we e ampli ied o con i m he p esence o HPV DNA and mouse β-globin was used as con ol. Amplicons leng hs we e con i med by aga ose gel elec opho esis. Only hemizygous emales we e used o he ansgenic mouse g oups in he expe imen . S udy Design Ten wild- ype (WT) (HPV16 -/- , G oup 1) and wel e ansgenic (HPV16 +/- , G oup 2) emale mice we e eu hanized a 24-26 weeks o age. La e , six HPV16 -/- (G oup 3) and six HPV16 +/- (G oup 4) emale mice we e sac i iced when 28-30 weeks-old. The mice we e humanely sac i iced by in ape i oneal pen oba bi al o e dose, ollowed by exsanguina ion h ough ca diac punc u e. Ches skin samples (app oxima ely 4 cm 2 ) we e collec ed o cell isola ion and low cy ome y analysis. Ma ched samples we e collec ed o his ological examina ion. His ology Skin samples we e ixa ed in 10% neu al bu e ed o malin o 48h. Samples we e dehyd a ed h ough g aded alcohols and xylene and pa a in-embedded in an au oma ic STP 120 p ocesso (Mic on, Boise, ID). 2 µm- hick sec ions we e s ained wi h haema oxylin-eosin (H&E) o his ological examina ion on ligh mic oscopy. Skin samples 23 we e classi ied as no mal skin, epide mal hype plasia o epide mal dysplasia. Mino dysplas ic oci on a hype plas ic backg ound we e also eco ded. P epa a ion o Single-Cell Suspensions Ches skin samples we e cleaned om excessi e blood essels and a issue and cu in o small pieces. These agmen s we e incuba ed o 2 hou s wi h 125 U/ml ype I collagenase (Gibco, Li e Technologies, Paisley, UK) in RPMI-1640 medium complemen ed wi h 1% glu amine, 1% penicillin-s ep omycin-ampho e icin B, 1% HEPES bu e (all om Sigma, S . Louis, MO) and 10% oe al bo ine se um (BioWes , Nuaillé, F ance) a 37 ºC and 150 pm in a 3031 o bi al incuba o (GFL, Bu gwedel, Ge many). The esul ing cell suspension was hen il e ed and cen i uged a 300  o 10 min a 4 ºC. Cells we e hen essuspended in phospha e bu e ed saline con aining 1% bo ine se um albumin and 20 mM sodium azide ollowed by ex acellula s aining. Immunopheno yping Following cell isola ion, he su ace pheno ype o he collec ed cells was assessed by low cy ome y using speci ic monoclonal an ibodies (mAb). To p e en non-speci ic an ibody binding, cells we e incuba ed wi h an i-mouse CD16/CD32 mAb o FcγR blocking. This was ollowed by incuba ion wi h an i-CD8 mAb phycoe y h in-cych ome 5- conjuga e (clone 53-6.7, BD Biosciences, San Diego, CA) and an i-CD107a mAb phycoe y h in-conjuga e (clone eBio1d4b, eBioscience, San Diego, CA). Following ex acellula s aining, he cells we e washed, ixed in 2% o maldehyde and washed wi h phospha e bu e ed saline con aining 1% bo ine se um albumin and 20 mM sodium azide. An ibody-labelled cells we e analysed in an EPICS XL low cy ome e using he EXPO32ADC so wa e (Beckman Coul e , Miami, FL). The collec ed da a iles we e analysed using he FlowJo so wa e 10.0.7 (FLOWJO, LLC, Ashland, OR). S a is ical Analysis S a is ical analyses we e execu ed in he G aphPad so wa e ( e sion 6.0, G aphPad So wa e, Inc. La Jolla, CA). S a is ical analysis be ween g oup pai s was pe o med using he Mann-Whi ney es . 24 Resul s T ansgenic Mice Show Epide mal Hype plasia and Dysplasia Analysis o skin samples showed ha he o ali y o WT mice (g oups 1 and 3) p esen ed no mal skin his ology (Fig. 2a) whils all ansgenic mice p esen ed skin lesions. In all 12 mice composing g oup 2 i was possible o obse e simple o papilla y, di use, a iably se e e epide mal hype plasia and papilloma osis wi h o hoke a o ic hype ke a osis (Fig. 2b). In lamma ion was mild, wi h a ew mac ophages, lymphocy es and mas cells p esen in he supe icial de mis. Also in his g oup, 2 animals (16.7%) p esen ed small epide mal dysplas ic oci. In g oup 4, 3 animals (50.0%) p esen ed di use epide mal dysplasia (Fig. 2c); o he 3 (50.0%) showed mul i ocal epide mal dysplasia in a hype plas ic backg ound. Sub-epide mal angiogenesis and de mal in lamma o y in il a es we e p ominen , showing nume ous mixed mononuclea leukocy es and neu ophils. Fig. 2: His opa hological changes induced by HPV16 oncogenes in FVB/n mice, H&E. a – WT animal; No mal skin his ology,  . b – 24-26 week-old HPV16 +/- animal; Epide mal hype plasia ex ending o he ollicula in undibulum and is hmus,  . c – 28-30 week-old HPV16 +/- animal; Epide mal dysplasia,  . No e enhanced sub-epide mal in lamma o y cell in il a ion. Inc eased CD8 + T lymphocy es numbe s and ac i a ion in HPV16 +/- mice In o de o assess he p esence o CD8 + T cells in HPV-associa ed lesions, lymphoid cells we e isola ed om ches skin issue and analysed by low cy ome y (Fig. 3a). As shown in Fig. 3b, ches skin samples om HPV16 +/- mice (g oups 2 and 4) p esen ed a signi ican ly highe pe cen age o CD8 + T cells when compa ed wi h hose o WT animals (g oups 1 and 3). Al hough he pe cen age o skin CD8 + T cells was sligh ly highe in g oup 4 han in g oup 2, i did no each s a is ical signi ican di e ence. In o de o de e mine i he CD8 + T cells ound in he skin o HPV16 +/- mice p esen ed e idence o cy o oxic ac i i y, he su ace exp ession o CD107a was e alua ed. In CD8 + T lymphocy es CD107a eaches he cell su ace when ly ic g anules su e exocy osis, hus exposing i s memb ane p o eins [144]. The e o e, his lysosome- associa ed memb ane p o ein is a commonly used su oga e ma ke o CTL deg anula ion [145]. As shown in Fig. 3c, he pe cen age o CD107a + CD8 + T cells in ches skin samples 25 o mice om g oups 2 and 4 we e ound signi ican ly highe when compa ed wi h espec i e con ols o g oups 1 and 3. This indica es ha in he HPV16 +/- mice skin in il a ing CTL eleased cy o oxic g anules. Mo eo e , in g oup 4 mice, 100% o which show mul i ocal o di use dysplasia, a ma kedly highe pe cen age o CD8 + T cells exp ess CD107a (P  0.001) han in g oup 2 mice, o which only 16.7% show ocal dysplas ic lesions. This esul shows a posi i e co ela ion in he p opo ions o ac i a ed CD8 + T cells and lesion se e i y. Fig. 3: Pe cen age o CD8 + and CD8 + CD107a + T cells wi hin o al lymphoid-ga ed cells ob ained om ches skin samples om WT and K14-HPV16 ansgenic mice. a – Rep esen a i e analysis o he ga ing s a egy employed. Numbe s wi hin g aphs co espond o he pe cen age o he ga ed popula ion. b – Pe cen ages o CD8 + T cells and c – pe cen ages o CD8 + CD107a + T cells in ga ed CD8 + T cells we e de e mined by low cy ome y a e skin issue collec ion and diges ion wi h collagenase. Ches skin samples we e collec ed om 24-26 weeks-old HPV -/- and HPV +/- mice (G oups 1 and 2, espec i ely) and 28-30 weeks-old HPV -/- and HPV +/- mice (G oups 3 and 4, espec i ely). G oup 1, n = 10; G oup 2, n = 12; G oup 3, n = 6; G oup 4, n = 6. Each do ep esen s an indi idual animal. Ba s ep esen he mean alue in each g oup. ** P  0.01; *** P  0.001. 32 low MHC I exp ession, CD8 + T cells play a decisi e ole in igh ing HPV-induced o opha yngeal cance s [155]. In he p esen wo k, we hypo hesize ha p aquiloside, he b acken oxin, exe s i s immunosupp essi e e ec by coun e ac ing he ac ion o CD8 + T cells agains papilloma i us-induced lesions. This would acili a e i al pe sis ence and he p og ession o ea ly HPV- and BPV-induced lesions, posing majo isks o human and animal heal h. In o de o es ha hypo hesis, we employed HPV16- ansgenic mice and analysed he e ec o p aquiloside on he popula ion o skin in il a ing CD8 + T cells. Ma e ial & Me hods Mice Cons uc ion o K14/HPV16 mice on a FVB/n backg ound has been p e iously epo ed [73]. These animals de elop cha ac e is ic mul i-s age cu aneous and u e ine ce ical ca cinogenesis, and we e gene ously dona ed by D s. Je ey A bei and Douglas Hanahan (Uni e si y o Cali o nia) h ough he USA Na ional Cance Ins i u e Mouse Reposi o y. The animal expe imen s we e app o ed by he Uni e sidade de T ás-os- Mon es e Al o Dou o e hics commi ee (10/2013) and he Po uguese Ve e ina y Di ec o a e (0421/000/000/2014). Animals we e main ained and b ed acco ding o Po uguese (Dec e o-Lei 113, Augus 7 h ) and Eu opean (EU Di ec i e 2010/63/EU) legisla ion, unde con olled condi ions o empe a u e (23 ± 2 ºC), ligh -da k cycle (12h ligh /12h da k) and ela i e humidi y (50 ± 10 %), using ha dwood bedding. Food and wa e we e p o ided ad libi um. Mice geno yping Animals we e geno yped a weaning, using ail ip samples as desc ibed p e iously [113, 114]. B ie ly, nucleic acids we e ex ac ed and DNA quali y and pu i y we e assessed. HPV16-E6 and -E2 genes we e ampli ied o con i m he in eg a ion o HPV DNA in o he mouse genome and a agmen o mouse β-globin was also ampli ied o con i m he quali y o he ex ac ed DNA. Leng hs o he agmen s we e con i med by aga ose gel elec opho esis. Only hemizygous emale mice we e used o he ansgenic mice g oups in he expe imen . P aquiloside isola ion P aquiloside was isola ed om b acken as p e iously desc ibed [156] wi h mino modi ica ions. B ie ly, 1000 g (d ied weigh ) b acken c osie s we e ha es ed a A cos de Valde ez, Po ugal, 41º 49´ 12´´ N, 8º 24´ 11´´ W) and a sample was deposi ed a he 33 Uni e sidade de T ás-os-Mon es e Al o Dou o he ba ium ( e e ence no. 18248). B acken was blended in dis illed wa e (10 L), s i ed a oom empe a u e o 1 hou and he ex ac was adso bed on 3 L o XAD-2 esin (Supelco, Sigma, S . Louis, MO). The esin was elu ed wi h me hanol (10 L) and he me hanol ex ac was concen a ed, dissol ed in wa e (400 ml) and ex ac ed wi h bu anol (5500 ml). The bu anol ex ac was ch oma og aphed on silica gel (Me ck, Kenilwo h, NJ). F ac ions con aining p aquiloside we e sepa a ed wice on oc adecyl-sylane silica gel (Fujy-Silysia, Kasugai Aichi, Japan) using me hanol-wa e mix u es o ob ain pu e p aquiloside. The compound was dis inguished om o he closely- ela ed b acken illudane oxins on he basis o i s cha ac e is ic 1 H and 13 C nuclea magne ic esonance (NMR) signals [157] using an A ance III 400 MHz spec ome e (B ucke , Bille ica, MA). Aliquo s we e p epa ed o each expe imen al week (7.5 mg), eeze-d ied and kep a -20ºC un il use. S udy Design Thi y ansgenic (HPV16 +/- ) and 15 wild- ype (WT, HPV16 -/- ) emale 18-20 weeks- old mice, showing di use cu aneous c us ing and papilloma osis we e employed. The animals we e sepa a ed in o h ee expe imen al g oups: g oup 1 (n = 15, HPV16 -/- mice), g oup 2 (n = 15, HPV +/- un ea ed mice) and g oup 3 (n = 15, HPV16 +/- mice ea ed o ally wi h 0.5 mg p aquiloside pe week, o 10 consecu i e weeks). The expe imen al animals we e moni o ed daily o signs o s ess o disease. All su i ing mice we e eu hanized a 28-30 weeks o age by an in ape i oneal pen oba bi al o e dose, ollowed by ca diac punc u e and exsanguina ion. Ches skin samples (app oxima ely 4 cm 2 ) we e collec ed o low cy ome y analysis. Ma ched samples we e collec ed o his ological examina ion. P aquiloside adminis a ion and oxici y Each week, a 7.5 mg p aquiloside aliquo was dissol ed in 300 µl e hanol (25 mg/mL) and he indi idual 0.5 mg dose (20 µl e hanol) was added o a s anda d whea cookie (Viei a, Vila No a de Famalicão, Po ugal) agmen weigh ing ca. 100 mg. Each dosed agmen was allowed o d y a oom empe a u e o 10 minu es and indi idually adminis e ed o a g oup 3 animal, in an indi idual emp y cage; he inges ion was isually moni o ed. In o de o con i m ha p aquiloside was indeed ac i e a he adminis e ed dosage, we looked o a lymphoid malignancy [128]. Thus, his ological analysis o kidney samples was used o con i m neoplas ic lymphoid cell in il a ion in his o gan. 34 Skin his ology Skin samples we e ixa ed in 10% neu al bu e ed o malin. Samples we e dehyd a ed h ough g aded alcohols and xylene and, pa a in embedded in an au oma ic STP 120 p ocesso (Mic on, Boise, ID). 2 µm- hick sec ions we e s ained wi h haema oxylin-eosin (H&E) o his ological examina ion on ligh mic oscopy. Skin samples we e classi ied as no mal skin, epide mal hype plasia, mul i ocal epide mal dysplasia in a hype plas ic backg ound and di use epide mal dysplasia. Isola ion o a Single-Cell Suspension Ches skin samples we e cleaned om excessi e blood essels and a issue and cu in o small pieces. This agmen s we e incuba ed o 2 hou s wi h 125 U/ml o ype I collagenase (Gibco, Li e Technologies, Paisley, UK) in RPMI-1640 medium complemen ed wi h 1% glu amine, 1% penicillin-s ep omycin-ampho e icin B, 1% HEPES bu e (all om Sigma) and 10% oe al bo ine se um (BioWes , Nuaillé, F ance) a 37 ºC and 150 pm in a 3031 shaking incuba o (GFL, Bu gwedel, Ge many). The esul ing cell suspension was hen il e ed and cen i uged a 300  o 10 min a 4 ºC. Cells we e hen essuspended in phospha e bu e ed saline con aining 1% bo ine se um albumin and 20 mM sodium azide ollowed by ex acellula s aining. Flow Cy ome y Analysis Following cell isola ion, he cellula immune pheno ype was assessed by low cy ome y using monoclonal an ibodies (mAb). Non-speci ic an ibody binding was p e en ed by incuba ing cells wi h an i-mouse CD16/CD32 mAb o FcγR blocking. This was ollowed by incuba ion wi h an i-CD8 phycoe y h in-cych ome 5-conjuga e (clone 53- 6.7, BD Biosciences, San Diego, CA), an i-CD107a (LAMP1) phycoe y h in-conjuga e (clone eBio1d4b) and an i-CD44 phycoe y h in-cych ome 7-conjuga e (clone IM7) mAb (bo h om eBioscience, San Diego, CA). Following ex acellula s aining, he cells we e washed, ixed in 2% o maldehyde and washed wi h phospha e bu e ed saline con aining 1% bo ine se um albumin and 20 mM sodium azide. An ibody-labelled cells we e analysed in an EPICS XL low cy ome e using he EXPO32ADC so wa e (Beckman Coul e , Miami, FL). The collec ed da a iles we e analysed using he FlowJo so wa e 10.0.7 (FLOWJO, LLC, Ashland, OR). S a is ical Analysis Flow cy ome y s a is ical analyses we e execu ed in he G aphPad so wa e ( e sion 6.0, G aphPad So wa e, Inc. La Jolla, CA). In column and do g aphs, each poin is ep esen a i e o an indi idual mouse and ba s ep esen he mean alue o he 35 espec i e g oup. Analysis be ween g oup pai s was pe o med using he Mann-Whi ney es . Kaplan-Meie su i al analysis coupled wi h a log ank es was pe o med using he PASW S a is ics so wa e ( e sion 18, IBM® SPSS®, Qua y Bay, Hong Kong). Resul s Gene al indings P aquiloside was isola ed a a 0.01% yield om b acken, as p e iously epo ed [128]. The compound's s uc u e (Fig. 4a) was con i med using NMR analysis and no o he illudane glycoside was de ec ed (Fig. 4b, c). T ansgenic animals showed cha ac e is ic di use cu aneous hype ke a osis and e y hema, oge he wi h a iably in ense p u i us, while WT mice showed no mal skin. All mice in g oups 1 and 2 su i ed he 10 weeks expe imen al pe iod. Ten mice (66.7%) om g oup 3 succumbed be o e he end o he s udy (Fig. 4d). His ological analysis o kidney samples showed no mal his ology in WT and un ea ed HPV +/- animals (Fig. 4e) and, mode a e o se e e, mul i ocal, pe i ascula in il a ion o lymphoblas ic cells, showing highly pleomo phic nuclei and up o 3 mi o ic igu es pe high-powe ield in 100% o p aquiloside- ea ed animals (g oup 3) (Fig. 4 ). 36 Fig. 4: P aquiloside and i s leukaemogenic e ec in K14-HPV16 mice. a – p aquiloside's s uc u al o mula. b and c – pa ial 2D NMR-HSQC spec a (ob ained in CD3OD) o p aquiloside. No e he co ela ions be ween ca bons and hei co esponding hyd ogens in he glucose esidue (b). No e in pa icula he co ela ions be ween ca bons 12 and 13 a 5.87 ppm and 10.86 ppm espec i ely, and hei co esponding hyd ogens in he cyclop opylidene ing (c). d – Kaplan-Meie su i al analysis. HPV +/- mice ea ed wi h p aquiloside show signi ican ly educed su i al (P  0.001) compa ed wi h un ea ed HPV -/- o HPV +/- animals. e – Un ea ed HPV +/- animal (g oup 2), showing no mal kidney his ology; H&E 200 . – P aquiloside-exposed HPV +/- animal (g oup 3) showing ypical p aquiloside- associa ed pe i ascula in il a ion by leukaemic lymphocy es. No e ma ked nuclea pleomo phism and mi o ic igu es; H&E 200 . 37 Cu aneous lesions The his ological analysis showed ha all g oup 1 mice (100.0%) p esen ed no mal skin his ology (Fig. 5a) whils all ansgenic mice (g oups 2 and 3) p esen ed skin lesions. In g oup 2, 3 animals (50.0%) p esen ed di use epide mal dysplasia (Fig. 5b); o he 3 (50.0%) showed epide mal hype plasia wi h mul i ocal dysplasia. Sub-epide mal angiogenesis and de mal in lamma o y in il a es we e p ominen in dysplas ic lesions, showing nume ous mixed mononuclea leukocy es and neu ophils. In g oup 3, he o ali y o mice (100.0%) showed di use epide mal dysplasia (Fig. 5c). Fig. 5: His opa hological changes induced by HPV16 oncogenes in FVB/n mice, H&E. a – g oup 1 animal. No mal skin his ology, 400 . b – g oup 2 animal. Epide mal hype plasia ex ending o he ollicula in undibulum, 400 . c – g oup 3 animal. Epide mal dysplasia, 400 . No e loss o ke a inocy ic pola i y and di e en ia ion. CD8 + T lymphocy es a e p esen in p aquiloside- ea ed and un ea ed animals In o de o de e mine he p opo ions o CD8 + T cells p esen in ches skin wi h o wi hou HPV-associa ed lesions, samples we e collec ed and cells isola ed, ollowed by low cy ome y analysis (Fig. 6a). As shown in Fig. 6b, g oup 2 and 3 (HPV +/- and p aquiloside- ea ed HPV +/- , espec i ely) mice showed signi ican ly highe pe cen ages o CD8 + T cells compa ed wi h HPV -/- mice (P  0.05). The pe cen age o CD8 + T cells in ches skin was no signi ican ly di e en be ween p aquiloside- ea ed and un ea ed HPV16 +/- mice (g oups 3 and 2, espec i ely). 38 Fig. 6: Pe cen age o CD8 + T cells wi hin o al lymphoid-ga ed cells ob ained om ches skin samples om WT and K14-HPV16 ansgenic mice. a – Rep esen a i e analysis o he ga ing s a egy u ilized. The numbe wi hin he g aph co esponds o he pe cen age o he ga ed popula ion. b – Pe cen ages o CD8 + T cells we e de e mined by low cy ome y a e skin issue collec ion and enzyma ic diges ion. Ches skin samples we e collec ed om 28-30 weeks-old HPV -/- (G oup 1), HPV +/- mice (G oup 2) and p aquiloside- ea ed (PTQ) HPV +/- mice (G oup 3). G oup 1, n = 5; G oup 2, n = 5; G oup 3, n = 5. Each do ep esen s an indi idual animal. Ba s ep esen he mean alue in each g oup. * P  0.05. P aquiloside dec eases he numbe o CD8 + CD107a + T cells in HPV16 +/- mice Nex , CD8 + T cells ound in skin samples we e analysed wi h he objec i e o de e mine i hey we e ac i ely deg anula ing, which indica es ongoing cy o oxic ac i i y. Fo ha pu pose, low cy ome y analysis was pe o med o assess he exp ession o su ace CD107a in hese cells (Fig. 7a). A signi ican ly highe pe cen age o CD8 + CD107a + T lymphocy es was obse ed in HPV +/- mice as compa ed wi h HPV -/- animals (P  0.01) (Fig. 7b). Addi ionally, HPV +/- mice exposed o p aquiloside also p esen ed a highe pe cen age o CD8 + CD107a + T lymphocy es when compa ed o WT animals, bu his was signi ican ly educed when compa ed o un ea ed HPV +/- mice (P  0.05). 39 Fig. 7: Pe cen age o CD8 + CD107a + T cells wi hin o al CD8 + T cells ob ained om ches skin samples om WT and K14-HPV16 ansgenic mice. a – Rep esen a i e analysis o he ga ing s a egy u ilized. Numbe s wi hin g aphs co espond o he pe cen age o he ga ed popula ion. b – Pe cen ages o CD8 + CD107a + T cells in ga ed CD8 + T cells we e de e mined by low cy ome y a e skin issue collec ion and enzyma ic diges ion. Ches skin samples we e collec ed om 28-30 weeks-old HPV -/- (G oup 1), HPV +/- mice (G oup 2) and p aquiloside- ea ed (PTQ) HPV +/- mice (G oup 3). G oup 1, n = 5; G oup 2, n = 5; G oup 3, n = 5. Each do ep esen s an indi idual animal. Ba s ep esen he mean alue in each g oup. *P 0.05; ** P  0.01. Reduced numbe o CD8 + CD44 + T cells in p aquiloside- ea ed ansgenic animals Ha ing de e mined ha CD8 + T cells p esen ed a cell su ace pheno ype associa ed wi h deg anula ion, exp ession o CD44, a ma ke indica ing a memo y cell pheno ype [158], was also assessed. As shown in Fig. 8 (a and b), a signi ican ly highe pe cen age o CD8 + CD44 + T lymphocy es was ound in HPV +/- mice as compa ed wi h HPV -/- animals (P  0.01). P aquiloside- ea ed HPV +/- mice p esen ed a signi ican ly educed pe cen age o CD8 + CD44 + T lymphocy es compa ed wi h un ea ed HPV +/- mice (P  0.05). In ac , g oup 3 did no show s a is ically di e en alues o g oup 1 (P  0.05). 40 Fig. 8: Pe cen age o CD8 + CD44 + T cells wi hin o al CD8 + T cells ob ained om ches skin samples om WT and K14-HPV16 ansgenic mice. a – Rep esen a i e analysis o he ga ing s a egy u ilized. Numbe s wi hin g aphs co espond o he pe cen age o he ga ed popula ion. b – Pe cen ages o CD8 + CD44 + T cells in ga ed CD8 + T cells we e de e mined by low cy ome y a e skin issue collec ion and enzyma ic diges ion. Ches skin samples we e collec ed om 28-30 weeks-old HPV -/- (G oup 1), HPV +/- mice (G oup 2) and p aquiloside- ea ed (PTQ) HPV +/- mice (G oup 3). G oup 1, n = 5; G oup 2, n = 5; G oup 3, n = 5. Each do ep esen s an indi idual animal. Ba s ep esen he mean alue in each g oup. * P  0.05;** P  0.01. Discussion CD8 + T lymphocy es a e impo an e ec o s o he cell-media ed immune esponse, especially agains i us-in ec ed o umou cells [117]. Upon ac i a ion hese cells may deg anula e, eleasing a se o ly ic enzymes capable o des oying a ge cells [159]. CD107a is a lysosome-associa ed memb ane p o ein which can be used as su oga e ma ke o deg anula ion [145]. In CD8 + T lymphocy es, CD107a eaches he cell su ace when ly ic g anules a e exocy osed, hus exposing i s memb ane p o eins [144]. As CD8 + T cells need o be ac i a ed in o de o elease cy o oxic g anules, he p esence o CD107a a he cell su ace is use ul o de e mine hei ac i a ion s a us [145]. Addi ionally, CD8 + T cells may also acqui e a memo y pheno ype, allowing a quicke ecall esponse, as memo y T cells can be mo e easily ac i a ed han nai e T cells [159]. CD44 is a cell-su ace glycop o ein and is cu en ly conside ed he bes ma ke o iden i y memo y CD8 + T cells [158]. Fu he mo e, CD44 is hough o ac as a egula o o he mo ili y o CTL in he umou mic oen i onmen [160]. 41 Al hough many papilloma i us (including some oncogenic HPV and BPV ypes) down- egula e MHC I exp ession as a s a egy o e ade immune su eillance media ed by CTL [154], hese cells s ill play a majo ole agains papilloma i us-induced lesions, namely in HPV-induced o opha yngeal cance s [155]. E ading he hos immune esponse is c i ical o main aining a long- e m in ec ion, allowing umou p og ession. In his con ex , en i onmen al (e.g. die a y) immunosupp essan p oduc s such as b acken and i s oxin, p aquiloside, may play an impo an ole. In his wo k, as expec ed, WT mice had no HPV-associa ed lesions, whils age- ma ched K14-HPV16 ansgenic mice showed hype plas ic lesions wi h dysplas ic oci o di use dysplasia. I is possible ha he di use dysplas ic lesions obse ed in p aquiloside- ea ed animals ep esen a mo e agg essi e s age in mul i-s ep ca cinogenesis compa ed wi h he o en ocal dysplasia obse ed in un ea ed animals. Howe e , he small numbe o animals ha su i ed he expe imen al pe iod and he absence o ob iously in asi e lesions do no allow us o i mly conclude ha p aquiloside enhanced umou p og ession. The inc easing agg essi eness o dysplas ic e sus hype plas ic lesions was accompanied by an inc eased bulk o in il a ing immune cells. These esul s indica e ha HPV-induced hype plas ic and dysplas ic skin lesions in K14-HPV16 ansgenic mice show inc eased in il a ion o CD8 + T cells when compa ed o no mal skin om age-ma ched WT mice. P aquiloside educed he CD8 + T cell in il a ion, al hough only sligh ly (P > 0.05). Ne e heless, p aquiloside did induce a signi ican educ ion in CD107a + and CD44 + CD8 + T cells. Al oge he , hese da a show ha cy o oxic T cells mig a e o he si e o HPV- induced lesions, a e ac i a ed o deg anula e and acqui e a memo y-pheno ype and, ha p aquiloside is capable o impai ing he unc ion o hese cells, explici ly by inhibi ing cellula ac i a ion and memo y pheno ype acquisi ion. The molecula mechanism behind his impai men should be an allu ing opic o u u e esea ch. G azing animals and human popula ions wo ldwide a e easily exposed o b acken and i s illudane oxins, including p aquiloside and o he ela ed compounds [123]. B acken is an abundan weed, especially in poo e pas u es, and p aquiloside accumula es in bo ine and o ine milk and mea [161-163], besides con amina ing unde g ound wa e s [164]. Mo eo e , some human popula ions in di e en coun ies consume b acken shoo s (known as c ozie s, b o o de samambaia in Po uguese o wa abi in Japanese) as pa o hei daily die [123]. In China, he p oduc ion o d y P e idium aquilinum a . la iusculum o ood uses (locally known as juecai) was es ima ed o in ol e app oxima ely 1000 companies in a business wo h 300 million USD pe annum [165]. This is conside ed he mos widely-consumed e n in China, wi h an es ima ed annual p oduc ion o 1200 ons in he Zhouzhou coun y, p o ince o Hunan. B acken and p aquiloside a e well-known 48 molecula ly [76], making K14-HPV16 mice a pa icula ly use ul model o HPV esea ch. Ma e ial & Me hods Animals Gene a ion o K14/HPV16 mice on a FVB/n backg ound has been p e iously epo ed [73]. K14-HPV16 ansgenic mice we e kindly dona ed by D s. Je ey A bei and Douglas Hanahan (Uni e si y o Cali o nia) h ough he USA Na ional Cance Ins i u e Mouse Reposi o y. The animal expe imen s we e app o ed by he Uni e sidade de T ás- os-Mon es e Al o Dou o E hics Commi ee (10/2013) and he Po uguese Gene al Ve e ina y Di ec o a e (app o al no. 0421/000/000/2014). Animals we e main ained and b ed acco ding o Po uguese (Dec e o-Lei 113, Augus 7 h ) and Eu opean (EU Di ec i e 2010/63/EU) legisla ion, unde con olled condi ions o empe a u e (23 ± 2 ºC), ligh -da k cycle (12h ligh /12h da k) and ela i e humidi y (50 ± 10 %), using co ncob bedding. Food and wa e we e p o ided ad libi um. Mice geno yping Animals we e geno yped a weaning, using ail ip samples as desc ibed p e iously [113, 114]. B ie ly, nucleic acids we e ex ac ed and DNA quali y and pu i y we e assessed. HPV16-E6 and -E2 genes we e ampli ied o con i m he p esence o HPV DNA and a agmen o mouse β-globin was also ampli ied as endogenous con ol. Leng hs o he amplicons we e con i med by aga ose gel elec opho esis. Only hemizygous emales we e used o he ansgenic mouse g oups in he expe imen . Expe imen al Design Fi y- ou 18 o 20 weeks-old emale mice we e di ided in o ou expe imen al g oups, acco ding o hei geno ype and aking in o conside a ion ha HPV ansgenes and celecoxib (CXB) adminis a ion could induce some mo ali y: g oup 1 (HPV16 -/- un ea ed animals, n = 12), g oup 2 (HPV16 +/- un ea ed animals, n = 12), g oup 3 (HPV16 -/- CXB- ea ed animals, n = 15) and g oup 4 (HPV16 +/- CXB- ea ed animals, n = 15). All su i ing mice we e humanely eu hanized a 24-26 weeks o age by in ape i oneal pen oba bi al o e dose, ollowed by exsanguina ion by ca diac punc u e. Ches skin samples (app oxima ely 4 cm 2 ) we e collec ed o low cy ome y analysis and ma ched skin samples we e collec ed o his ological analysis. 49 Celecoxib adminis a ion Celecoxib (P ize , New Yo k, NY) was dissol ed in d inking wa e a a concen a ion o 0.5 mg/ml, es ima ing an a e age daily wa e in ake o 5.0ml pe mouse, and a dose o 46.7 mg/kg/day and 2.5 mg/animal/day in an a e age mouse weigh ing 30 g. This is a well- ole a ed mode a e dose, as shown in p e ious assays [177]. Howe e , K14-HPV16 animals d ama ically inc eased hei wa e in ake when CXB was added (possibly due o he highly pala able lac ose p esen in he ehicle) eaching up o 15 ml pe animal. The CXB concen a ion was hus educed om he s a o he hi d week onwa ds down o 0.2 mg/ml, esul ing in a dec ease in consump ion and an e ec i e dose o 93 mg/kg/day and 2.8 mg/animal/day. The a e age dose du ing he o e all expe imen al pe iod was hus 124 mg/kg/day and 3.72 mg/animal/day. His ological Analysis Skin samples we e ixa ed in 10% neu al bu e ed o malin o 48 hou s. Samples we e dehyd a ed h ough g aded alcohols and xylene and pa a in embedded in an au oma ic STP 120 p ocesso (Mic on, Boise, ID). 2 µm- hick sec ions we e s ained wi h haema oxylin-eosin (H&E) o his ological e alua ion on ligh mic oscopy. Skin samples we e classi ied as no mal skin, epide mal hype plasia and epide mal dysplasia. P epa a ion o Single-Cell Suspensions Ches skin samples we e cu in o small pieces a e excessi e blood essels and a issue emo al. Skin agmen s we e incuba ed o 2 hou s wi h 125 U/ml ype I collagenase (Gibco, Li e Technologies, Paisley, UK) in RPMI-1640 medium complemen ed wi h 1% glu amine, 1% penicillin-s ep omycin-ampho e icin B, 1% HEPES bu e (all om Sigma, S . Louis, MO) and 10% oe al bo ine se um (BioWes , Nuaillé, F ance) a 37 ºC and 150 pm in a 3031 o bi al incuba o (GFL, Bu gwedel, Ge many). Subsequen ly, he esul ing cell suspension was il e ed and cen i uged a 300  o 10 min a 4 ºC. Cells we e essuspended in phospha e bu e ed saline con aining 1% bo ine se um albumin and 20 mM sodium azide ollowed by low cy ome y analysis. Immunopheno yping Following cell isola ion, he su ace pheno ype o he collec ed cells was assessed by low cy ome y using speci ic monoclonal an ibodies (mAb). Cells we e incuba ed wi h an i-mouse CD16/CD32 mAb o FcγR blocking, o p e en non-speci ic an ibody binding. Nex , cells we e incuba ed wi h an i-CD8 mAb phycoe y h in-cych ome 5-conjuga e (clone 53-6.7, BD Biosciences, San Diego, CA) and an i-CD107a (LAMP1) mAb phycoe y h in- 50 conjuga e (clone eBio1d4b, eBioscience, San Diego, CA). Following ex acellula s aining, he cells we e washed, ixed in 2% o maldehyde and washed wi h phospha e bu e ed saline con aining 1% bo ine se um albumin and 20 mM sodium azide. An ibody-labelled cells we e analysed in an EPICS XL low cy ome e using he EXPO32ADC so wa e (Beckman Coul e , Miami, FL). The assembled da a iles we e analysed using he FlowJo so wa e 10.0.7 (FLOWJO, LLC, Ashland, OR). S a is ical Analysis S a is ical analyses we e pe o med using he G aphPad so wa e ( e sion 6.0, G aphPad So wa e, Inc. La Jolla, CA). In column and do g aphs each poin ep esen s indi idual mice, while ba s ep esen he mean o he espec i e g oup. S a is ical analysis be ween g oup pai s was done using he Mann-Whi ney es . Resul s Gene al indings All K14-HPV16 mice showed ypical cu aneous changes, including di use hype ke a osis and e y hema. Celecoxib- ea ed HPV +/- mice (g oup 4) showed signi ican mo ali y: 10 ou o 15 mice (66.7%) succumbed be o e he end o he s udy. All mice in g oups 1, 2 and 3 su i ed un il he end o he s udy. His ological analysis His ological analysis o skin samples (Table 2) showed ha all WT mice, g oups 1 and 3, p esen ed no mal skin his ology (Fig. 9a). In g oups 2 and 4 (un ea ed and CXB- ea ed HPV +/- mice, espec i ely), he o ali y o mice showed simple o papilla y, di use, a iably se e e epide mal hype plasia ex ending o he ollicula in undibula and papilloma osis wi h o hoke a o ic hype ke a osis (Fig. 9b). Addi ionally, 2 animals (16.7%) om g oup 2 p esen ed mul i ocal epide mal dysplas ic oci wi h pa ake a o ic hype ke a osis wi hin he hype plas ic backg ound (Fig. 9c). The e we e signs o mild in lamma ion, wi h a small amoun o mac ophages, mas cells and lymphocy es in he supe icial de mis. Dysplas ic oci we e associa ed wi h inc eased numbe s o leukocy es in il a ing he de mo-epide mic junc ion and wi h in ense angiogenesis. 51 Table 2: His ological classi ica ion o HPV16-induced skin lesions om 24-26 weeks-old emale mice. Celecoxib – CXB. G oup Cu aneous lesions Incidence (%) No mal skin Epide mal hype plasia Epide mal 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: His opa hological changes induced by HPV16 oncogenes in FVB/n mice, H&E. a – WT animal; No mal skin his ology, 100 . b – CXB- ea ed HPV16 +/- animal; Epide mal hype plasia ex ending o he ollicula in undibulum and is hmus, 100 . c – Un ea ed HPV16 +/- mouse; Epide mal dysplasia, 200 . No e ma ked pa ake a o ic hype ke a osis, loss o cell pola i y, enhanced anisoka yosis and mi o ic ac i i y. The de mal-epide mal junc ion is obscu ed by se e e in lamma o y cell in il a ion. CTL in il a ion and ac i a ion in celecoxib- ea ed mice Wi h he pu pose o s udy CD8 + T cell in il a ion in HPV-induced lesions, lymphoid cells om mice ches skin issue we e isola ed and a low cy ome y analysis o he eco e ed cells was pe o med (Fig. 10a). Skin samples om un ea ed HPV +/- mice showed a signi ican ly highe pe cen age o CD8 + T cells compa ed wi h un ea ed WT animals (P  0.01) (Fig. 10b). Also, al hough s a is ical signi icance was no achie ed, CXB- ea ed WT mice showed dec eased CD8 + T cell in il a ion when compa ed o CXB- ea ed HPV +/- animals and he same was obse ed be ween samples om WT mice ea ed wi h CXB (g oup 3) and un ea ed WT animals (g oup 1) (Fig. 10b). Mo eo e , HPV +/- mice ea ed wi h CXB (g oup 4) showed less CD8 + T lymphocy es compa ed wi h un ea ed ansgenic animals (g oup 2) (P  0.01) (Fig. 10b). Nex , he objec i e was o s udy whe he he CD8 + T lymphocy es ound in mouse skin samples showed signs o cy o oxic ac i i y (Fig. 10a). Thus, exp ession o CD107a (LAMP1) a he cell su ace was assessed by low cy ome y. CD107a eaches he su ace o CTL when ly ic g anules a e eleased, exposing hei memb ane p o eins du ing 52 exocy osis [144]. Thus, his lysosome-associa ed memb ane p o ein is equen ly used as an immunological ma ke o CTL deg anula ion [145]. Fig. 10c shows a signi ican ly highe pe cen age o CD8 + CD107a + cells in samples om HPV +/- mice ea ed wi h CXB (g oup 4) when compa ed wi h un ea ed HPV +/- (g oup 2) (P  0.01). Fig. 10: Pe cen age o CD8 + and CD8 + CD107a + T cells wi hin o al lymphoid-ga ed cells ob ained om ches skin samples om WT and K14-HPV16 ansgenic mice. a – Rep esen a i e analysis o he ga ing s a egy employed. Numbe s wi hin g aphs co espond o he pe cen age o he ga ed popula ion. b – Pe cen ages o CD8 + T cells and c – pe cen ages o CD8 + CD107a + T cells in ga ed CD8 + T cells we e de e mined by low cy ome y a e skin issue collec ion and diges ion wi h collagenase. Ches skin samples we e collec ed om WT and HPV +/- mice (G oups 1 and 2, espec i ely) and CXB- ea ed (CXB) WT and HPV +/- mice (G oups 3 and 4, espec i ely) a 24-26 weeks o age. G oup 1, n = 5; G oup 2, n = 5; G oup 3, n = 5; G oup 4, n = 5. Each do ep esen s an indi idual animal. Ba s ep esen he mean alue in each g oup. ** P  0.01. 53 Discussion The de elopmen o HPV-associa ed malignancies depends on a pe sis en HPV in ec ion. Ul ima ely, he abili y o he immune sys em o elimina e he i us is he key elemen o decide whe he a HPV in ec ion is clea ed o e ol es o cance [2]. HPV p esen s well-known mechanisms o e ade hos immuni y and delay i s elimina ion, hus acili a ing i al pe sis ence [154]. When he i us is de ec ed, an inna e immune esponse occu s, and leads o he de elopmen o an adap i e immune esponse. A undamen al pa o his adap i e esponse is cell-media ed immuni y, cha ac e ized by he ac i i y o a as numbe o CD8 + and CD4 + T lymphocy es [39]. In ac , in il a ion by hese cells in HPV-associa ed lesions has al eady been shown o lead o eg ession [148, 178]. CTL in il a ion d i es he esponse induced by an expe imen al he apeu ic accine in pa ien s wi h ce ical in aepi helial lesions [171] and co ela es wi h a be e p ognosis in pa ien s wi h HPV-posi i e head and neck cance [155, 172, 173]. Ch onic in lamma ion is a key ea u e associa ed wi h ca cinogenesis, namely in he case o HPV in ec ion [8]. COX-2 is an enzyme wi h an impe a i e ole in he me abolism o a achidonic acid, which leads o he p oduc ion o p os aglandins, which in u n p omo e in lamma ion [129]. In ac , COX-2 is o e -exp essed in se e al malignancies, including ce ical cance [130-134]. As in lamma ion is known o con ibu e o cance p og ession [8], COX-2 inhibi ion is expec ed o esul in umou g ow h inhibi ion whils educing in lamma ion. Some non-s e oidal an i-in lamma o y d ugs, like aspi in and ibup o en, ha e al eady shown p omising esul s as an i- umou he apy in bo h pa ien s and p e-clinical animal models [179-182]. Howe e , hese d ugs ha e e y low speci ici y. Selec i e COX-2 inhibi o s such as CXB and o ecoxib ha e al eady been used o p e en he de elopmen o colo ec al adenomas [183, 184]. Fu he mo e, his COX-2 inhibi o has been showing p omising esul s in a umou model o human colon cance when combined wi h chemo he apeu ic d ugs [185]. In his s udy, he e ec o he selec i e COX-2 inhibi o CXB o e he a icking and ac i a ion o CD8 + T lymphocy es was examined in he K14-HPV16 mouse model, a p ope model o s udy HPV-induced ca cinogenesis due o i s g ea simila i ies wi h he human clinical disease [76]. High mo ali y was obse ed among CXB- ea ed animals, p esumably due o CXB- ela ed oxici y. The esul s p esen ed he ein show ha CXB educes he numbe o umou - in il a ing CD8 + T cells when compa ed wi h un ea ed mice. S ill, despi e he dec ease in cell numbe s, CXB- ea ed mice ha e a highe pe cen age o ac i a ed and deg anula ing CTL compa ed wi h un ea ed animals. These indings sugges ha CXB educes he numbe o umou -in il a ing CD8 + T cells while enhancing hei e ec o unc ions. These 54 da a a e in ag eemen wi h a p e ious s udy using glioma-bea ing mice whe e COX2 -/- mice had inc easing pe cen ages o umou -in il a ing CD8 + CD107a + lymphocy es [175]. This may be explained by ano he s udy epo ing ha COX-2 ac i i y leads o CD8 + T cell senescence and his ajec o y may be opposed by COX-2 inhibi ion, as shown by inc eased le els o CD28 and in e leukin-2 in CD8 + T cells [174]. Fu he mo e, hype plas ic epide mal lesions we e e iden in all su i ing ansgenic mice, whils all WT animals p esen ed no mal skin his ology, as expec ed. Mul i ocal dysplas ic epide mal lesions we e es ic ed o un ea ed HPV +/- animals. These indings sugges ha CXB blocked umou p og ession a he hype plas ic s age, bu he small numbe o dysplas ic lesions obse ed does no allow o any de ini i e conclusions. In ac , COX-2 inhibi ion boos ed he e icacy o a DNA accine exp essing he HPV E7 oncogene, by enhancing umou -in il a ing CD8 + T cells and slowing umou g ow h [186]. Howe e , his s udy was pe o med in mice bea ing allog a ed TC1 lung cells immo alized by he HPV16 E6 and E7 oncogenes and ans o med by he c-Ha- as oncogene. Compa isons be ween his model and K14-HPV16 mice a e limi ed, because allog a s do no ep oduce HPV-associa ed mul i-s ep ca cinogenesis, being di ec ly implan ed in he subcu is wi h a ully malignan pheno ype. Also, ke a inocy es and no lung cells a e he a ge s o papilloma i us in ec ion. I emains unclea whe he CTL a e ac i a ed a egional lymph nodes o a he lesion loca ion and his would be an in e es ing poin o add ess in he u u e. The esul s p esen ed he ein sugges ha CXB induces augmen ed CTL deg anula ion in HPV- induced lesions, possibly con ibu ing o p e en malignan p og ession in his animal model. Conclusion The p esen da a con i m he po en ial o CXB o enhance deg anula ion by umou -in il a ing CD8 + T lymphocy es. Mo eo e , he e ec o CXB seems o be mo e complex han p e iously hough , as i also educed he o e all numbe o umou - in il a ing CD8 + T cells. Fu u e s udies add essing he impac o COX-2 inhibi o s on he p ognosis o pa ien s bea ing HPV-induced lesions should ake in o accoun bo h he numbe o in il a ing CD8 + T cells and hei ac i a ion s a us. 55 Chap e 6: Gene al Discussion and Conclusions 56 57 HPV-associa ed malignancies a e a genuine heal h p oblem wo ldwide, wi h emphasis on he inc ease in he numbe o HPV-associa ed o opha yngeal ca cinomas [152]. P ophylac ic accina ion emains he mos e ec i e me hod o con ol HPV in ec ions. Cu en ly, he e a e wo e ec i e comme cialized accines: Ce a ix™ (GlaxoSmi hKline Biologicals, Rixensa , Belgium), a bi alen HPV16/18 accine; and Ga dasil™ (Me ck Vaccines, Wes Poin , PA), a quad i alen HPV6/11/16/18 accine. Bo h a e HPV L1 i us-like pa icles (VLP) accines and hey ac by boos ing he p oduc ion o neu alising an ibodies di ec ed agains he L1 capsid p o ein [187]. Despi e hei e ec i eness, hese accines p esen a ew limi a ions, as he i us- ype es ic ion. The i s is es ic ed o wo HPV ypes (bi alen ) and he second is es ic ed o ou ypes (quad i alen ). Looking o inc ease ype-speci ic p o ec ion, Me ck managed o c ea e he nona alen HPV L1 VLP accine by adding he VLPs om i e oncogenic HPV ypes (31, 33, 45, 52 and 58) in addi ion o he ou ypes used in he quad i alen accine. This esul ed in a 20% inc ease in p o ec ion, in addi ion o he 70% ob ained om he quad i alen accine, in a o al o app oxima ely 90% p o ec ion agains ce i al cance [188]. Howe e , despi e hese ad ances in disease p e en ion, an e ec i e he apeu ic accine agains HPV-induced malignancies emains absen . In o de o de elop a he apeu ic s a egy, i is necessa y o ga he a la ge amoun o in o ma ion on he ea u es o disease p og ession as well as o he e en s occu ing in he lesions mic oen i onmen . I is accep ed ha he ca cinogenesis p ocess induced by a HPV in ec ion is ma ked by he exis ence o a p ominen immune esponse [11, 18]. Thus, a g ea deal o cellula popula ions and chemical ac o s a e p esen and play a speci ic ole in ca cinogenesis, ei he p omo ing o impai ing i . A he end, i should be he balance be ween all p o- and an i- umou s imuli ha will decide whe he he i al in ec ion is clea ed o is allowed o p og ess owa ds malignancy. The e o e, a he apeu ic accine could possible enhance he ac ion o an i- umou e ec o s and/o inhibi he unc ion o known p o- umou media o s. Cy o oxic CD8 + T lymphocy es, o ins ance, a e he main cells associa ed wi h he elimina ion o i us-in ec ed and ans o med cells. These cells a e pa o he adap i e immune esponse and depend on an igen p esen a ion by MHC I molecules and o he co- s imula o y signals o become ac i a ed and exe hei unc ions [116, 117]. Howe e , du ing he cou se o HPV in ec ion, exp ession o MHC I molecules is usually down- egula ed. This is one o he mos common immune e asion mechanisms employed by he HPV E5 oncop o ein [154]. 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