Strategies for cancer immunotherapy using induced pluripotency stem cells-based vaccines

cance s Re iew S a egies o Cance Immuno he apy Using Induced Plu ipo ency S em Cells-Based Vaccines B uno Be na des de Jesus 1,†, B uno Miguel Ne es 1,†, Manuela Fe ei a 2,3,* and Sand ina Nób ega-Pe ei a 1,4,* 1Depa men o Medical Sciences and Ins i u e o Biomedicine—iBiMED, Uni e si y o A ei o, 3810-193 A ei o, Po ugal; b [email p o ec ed] (B.B.d.J.); [email p o ec ed] (B.M.N.) 2Cen e o Neu oscience and Cell Biology (CNC), Uni e si y o Coimb a, UC Bio ech, Biocan Pa k, 3060-197 Can anhede, Po ugal 3Champalimaud Resea ch, Champalimaud Cen e o he Unknown, Champalimaud Founda ion, 1400-038 Lisboa, Po ugal 4Ins i u o de Medicina Molecula João Lobo An unes, Faculdade de Medicina, Uni e sidade de Lisboa, A . P o esso Egas Moniz, 1649-028 Lisboa, Po ugal *Co espondence: [email p o ec ed] (M.F.); sand ina.pe [email p o ec ed] (S.N.-P.) †These au ho s con ibu ed equally o his wo k. Recei ed: 31 Oc obe 2020; Accep ed: 27 No embe 2020; Published: 30 No embe 2020   Simple Summa y: E ec i e cance immuno he apies, wi h he objec i e o boos umo -speci ic immune esponses, is a game-change in pe sonalized cance ea men . In immuno he apy, he immune sys em is exploi ed o ecognize and des oy cance cells, and his is only possible i a ull ecapi ula ion o umo speci ic an igens complexi y is achie ed. Pa ien -de i ed induced plu ipo en s em cells (iPSCs) sha e se e al cha ac e is ics wi h cance (s em) cells (CSCs). The exploi a ion o iPSCs as a sou ce o umo - and pa ien -speci ic an igens guiding he immune sys em agains cance has been add essed ecen ly in mice. He e, we will deba e no el indings on he po en ial implica ion o cellula ep og amming and iPSCs plas ici y o he design o no el cance immuno he apeu ic s a egies. Abs ac : Despi e imp o emen s in cance he apy, me as a ic solid umo s emain la gely incu able. Immuno he apy has eme ged as a pionee ing and p omising app oach o cance he apy and managemen , and in pa icula in ended o ad anced umo s un esponsi e o cu en he apeu ics. In cance immuno he apy, componen s o he immune sys em a e exploi ed o elimina e cance cells and ea pa ien s. The ecen clinical successes o immune checkpoin blockade and chime ic an igen ecep o T cell he apies ep esen a u ning poin in cance ea men . Despi e hei po en ial success, cu en app oaches depend on e icien umo an igen p esen a ion which a e o en inaccessible, and mos umo s u n e ac o y o cu en immuno he apy. Pa ien -de i ed induced plu ipo en s em cells (iPSCs) ha e been shown o sha e se e al cha ac e is ics wi h cance (s em) cells (CSCs), elici ing a speci ic an i- umo al esponse when injec ed in oden cance models. Indeed, a i icial cellula ep og amming has been widely compa ed o he biogenesis o CSCs. He e, we will discuss he s a e-o - he-a on he po en ial implica ion o cellula ep og amming and iPSCs o he design o pa ien -speci ic immuno he apeu ic s a egies, deba ing he simila i ies be ween iPSCs and cance cells and in oducing po en ial s a egies ha could enhance he e iciency and he apeu ic po en ial o iPSCs-based cance accines. Keywo ds: iPSCs; cance ; ep og amming; immuno he apy; neoan igens; accines Cance s 2020,12, 3581; doi:10.3390/cance s12123581 www.mdpi.com/jou nal/cance s Cance s 2020,12, 3581 2 o 25 1. In oduc ion Me as a ic solid umo s emain an un esol ed clinical challenge and one o he leading causes o dea h. Tumo immunosu eillance is widely ecognized as an impo an mechanism o educe cance incidence by he elimina ion o p ecance ous lesions. Tumo es ablishmen and p og ession in ol e he p oli e a ion o hypo-immunogenic cells ha manage o e ade he immune sys em by exploi ing immune checkpoin s, such as he p og amed cell dea h 1 (PD1) PD-ligand 1 (PDL1) axis and cy o oxic T lymphocy e an igen-4 (CTLA-4) [1]. The use o he pa ien ’s immune sys em o elimina e neoplas ic cells has long been pos ula ed [ 2 ]. Howe e , only he ecen inc easing unde s anding o how cance in e ac s wi h he immune sys em has allowed he p oposal o success ul cance immuno he apeu ic app oaches, c ea ing widesp ead in e es and high expec a ions in he ield [ 3 – 5 ]. Immuno he apeu ic s a egies ha success ully ain he immune sys em o e icien ly a ge umo s include (1) unlocking immune checkpoin s o achie e umo ecogni ion and (2) p iming he immune sys em o use a wide epe oi e o an igens p esen ed on umo cells. The la e includes chime ic an igen ecep o s (CAR) T-cell he apy, whe e T cells a e gene ically enginee ed o p oduce a i icial T-cell ecep o s, showing imp essi e clinical ou comes in ea ing pa ien s wi h elapsed o e ac o y B cell malignancies [ 6 – 8 ]. Al hough CAR-T cells ha e been success ully used e en in umo s un esponsi e o con en ional he apies, many ques ions limi ing i s clinical applica ion s ill need o be add essed. Fo ins ance, CAR-T he apies may lead o acu e cy okine elease synd ome, issues ela ed wi h long- e m sa e y, acquisi ion o esis ance and lack o clinical e icacy in solid umo s, issues ha need o be e ined [ 9 , 10 ]. Mo eo e , se e al issues pe sis wi h he usage o T-cells, including loss o umo an igen exp ession, o - a ge oxici y and he possibili y o in i o ans o ma ion, amongs o he s [8,11,12]. E icien umo an igen p esen a ion and s imula o y signals by an igen-p esen ing cells (APCs) is undamen al o a sus ained ac i a ion o cy o oxic T and B-cells, and c ucial o he success o any immuno he apeu ic app oach [ 3 , 13 – 16 ]. Indeed, many umo s p esen educed sel -an igen p esen a ion due o dec eased exp ession o majo his ocompa ibili y complex (MHC)-class 1 molecules, which esul s om he s ong selec i e p essu e imposed on umo cells in o de o e ade immuni y du ing ea ly umo igenesis [ 17 ]. Cance he e ogenei y, and he p esence o undi e en ia ed cance (s em) cells (CSCs), imposes addi ional ba ie s wi h he selec i e exp ession o an igens by di e en cance cell popula ions wi hin a umo [ 18 ]. Al hough clonal neo-an igen bu den is associa ed wi h imp o ed esponse o immuno he apy, i is cu en ly challenging o design imp o ed immuno he apeu ic app oach as umo samples a e o en inaccessible [ 19 ]. Addi ionally, no el mu a ions o clonal selec ion may lead o he esis ance o cu en immuno he apeu ic s a egies a ge ing unique an igens. Thus, a majo challenge o cance immuno he apy is he need o de elop mo e e ec i e s a egies since, o da e, immuno he apy only wo ks in a subse o cance s and only a ac ion o pa ien s wi h cance espond o immuno he apy He e, we will un eil he po en ial applica ion o cellula ep og amming and induced plu ipo en s em cells (iPSCs)-based cance accines as no el s a egies o ackle umo he e ogenei y. Al hough ou o scope o he p esen e iew, i is wo h o men ion ha besides di ec use o iPSCs as a no el sou ce o cance an igens, g ea e o s ha e been pu on he ep og amming o iPSCs in o umo -speci ic T cell, dend i ic cells (DCs), imma u e na u al-kille cells (iNKs,) and gama-del a T cells ( γδ T) ha a e hen applied in adop i e an i umo immuno he apies [20–28]. 2. Cellula Rep og amming Is P one o E o s In 2006, Yamanaka and colleagues challenged he cell di e en ia ion dogma, by demons a ing ha a combina ion o he ou ansc ip ion ac o s (TFs) Oc 3/4, Sox2, Kl 4, and cMyc we e enough o ep og am di e en ia ed mouse e al and adul ib oblas s in o iPSCs [ 29 ], and one yea la e , human iPSCs we e gene a ed [ 30 , 31 ]. These cells showed cha ac e is ics o emb yonic s em cells (ESCs) including mo phology, ESC-speci ic gene exp ession and he abili y o o m all h ee emb yonic ge m laye s as depic ed by e a oma o ma ion in SCID mice and con ibu ion o chime ic mice [ 29 ]. Despi e Cance s 2020,12, 3581 3 o 25 being a majo hallma k in cell biology and p o iding unp eceden ed oppo uni y o cell he apy, se e al challenges s ill pe sis ha limi hei cu en applica ions, including hei inhe en p ope ies o umo igenici y, immunogenici y, and he e ogenei y [32]. iPSCs ep og amming is a e y ine icien p ocess leading o a he e ogeneous popula ion o cells ei he ailing o eaching only pa ial ep og amming [ 33 ]. Addi ionally, i is no uncommon ha ep og amed iPSCs p esen mixed esponse o di e en ia ion s imuli, jeopa dizing hei po en ial applica ion [ 34 – 36 ]. Bo h gene ic and epigene ic a iabili y may con ibu e o iPSCs he e ogenei y [ 37 ], as well as ecu en gene ic abe a ions [ 35 , 38 – 41 ]. Amongs he epigene ic ailu es is he lack o capaci y o ully ese hei soma ic memo y, o o ully es ablish a new soma ic iden i y [ 34 , 36 , 42 – 44 ]. Suppo ing his obse a ion is he common de ec ion o imp in ing de ec s in iPSCs, some po en ially hal ing iPSCs cha ac e is ics [ 41 , 45 – 50 ]. Rega ding he gene ic al e a ions, iPSCs may e lec he dono gene ic backg ound. Indeed, i has been demons a ed ha dono gene ic ma e ial accoun ed o mo e unc ional di e ences be ween iPSCs, han he cell ype [ 51 ]. Indeed, Kilpinen and colleagues [ 52 ] desc ibed he sys ema ic gene a ion o 711 iPSC clones de i ed om 301 heal hy indi iduals, ou lining ha 5–46% o he a ia ion be ween di e en iPSCs pheno ypes a ise om di e ences be ween indi iduals. Al hough some o he speci ic ai s we e associa ed wi h plu ipo ency, o he s we e po en ially associa ed wi h pa hology and umo igenici y. Mo e ecen ly Salah Mahmoudi and colleagues [ 53 ] show ha ib oblas om old mice exhibi inc eased a iabili y du ing ep og amming. By pe o ming mul i-omics p o iling o ib oblas cul u es om young and old mice, Mahmoudidi e ealed he exis ence o ’ac i a ed ib oblas s’ ha sec e e in lamma o y cy okines, mo e p one o ep og amming. O he sou ces o he e ogenei y include he selec ion o clonal iPSCs ca ying speci ic mu a ions o he acquisi ion o mu a ions du ing he ep og amming p ocess. iPSCs de i a ion s a s om a ew cells and a iabili y was shown o be ela ed wi h p e-exis ing a ia ion in he o iginal soma ic cells, ei he con ibu ing o highe o lowe ep og amming a es [ 38 , 54 – 59 ]. Se e al pa ien -speci ic soma ic mu a ions impac on iPSCs ep og amming [ 60 ], aising impo an issues when pa ien -de i ed iPSCs a e equi ed. Those include dys unc ion o he mi ochond ial espi a o y chain [ 61 , 62 ] o ch omosomal abno mali ies [ 63 , 64 ]. Fu he mo e, du ing he in i o ep og amming p ocess, iPSCs may acqui e (addi ional) soma ic mu a ions which could ela e o he ep og amming s a egy. iPSCs de i a ion does no only co ela es wi h p e-exis ing mu a ions bu could also lead o “de no o” mu a ions [ 51 , 65 ]; due o he complexi y o he ep og amming p ocess is was e i ied ha iPSCs acqui e mo e poin mu a ion han ESCs. One example is he oxida i e s ess linked mu a ions acqui ed du ing cellula ep og amming [66,67]. As men ioned, he ep og amming p ocess o he subsequen expansion in cul u e ep esen addi ional sou ce o iPSCs he e ogenici y. I was p e iously shown ha 17 di e en human ESCs lines main ained in di e en labo a o ies p esen 843 copy numbe a ia ions (CNV) [ 68 ]. In e es ingly, se e al o he genes de ec ed wi hin CNV si es had al e ed exp ession and we e unc ionally linked o cance . These esul s suppo he hypo hesis ha cellula he e ogenei y may lead o he acquisi ion o cance like p ope ies. One example linking iPSCs o cance biogenesis is he abno mal exp ession o TERT, which ac i i y is mainly es ic ed o s em cells in he adul issues [ 69 – 71 ]. As iPSCs can be gene a ed om soma ic cells, hey show g ea po en ial o de eloping pa ien speci ic disease modeling, which can be used o s udy he unde lying mechanisms and no el ea men s o cance [ 72 ]. Simila ly, in i o ep og amming is also p one o he e ogenei y. In i o iPSCs ha e an unp eceden ed capaci y o o m emb yo-like s uc u es, including he h ee ge m laye s o he emb yo and ex aemb yonic issues, s ill only a small pe cen age o cells could gi e ise o such s uc u es [ 73 ]. O no ewo hy, and using an assay whe e GFP-exp essing iPSCs and ESCs we e mic oinjec ed in o he mo ula and he esul ing blas ocys s we e examined, Abad e al obse ed ha iPSCs gene a ed in i o con ibu ed o he ophec ode m wi h a ema kable e iciency (56%), which was in con as o ESCs (0%) [73]. Cance s 2020,12, 3581 4 o 25 iPSCs Sha e Simila i ies wi h Cance S em Cells CSCs a e subpopula ions o cance cells wi h s em cell ea u es as sel - enewal abili y and mul i-lineage di e en ia ion ha d i e umo g ow h and he e ogenei y. Impo an ly, CSCs ha e been in oked as he main d i e s o me as a ic dissemina ion, elapse and he apeu ic esis ance [ 74 , 75 ]. Malignan and me as a ic umo s, which e ade immune clea ance, show a highe incidence o cance -speci ic soma ic mi ochond ial DNA (m DNA) mu a ions in p o ein-coding o RNA-coding egions [ 76 ] and exp ession o s emness genes [ 75 ], sugges ing ha lowe immune clea ance is associa ed wi h inc ease cance -speci ic mu a ions and exp ession o s emness ma ke s. Simila ly, o ESCs, iPSCs also sha e a numbe o cha ac e is ics wi h CSCs including sel - enewal and p oli e a ion, exp ession o s em cell ma ke s and al e ed me abolism. The simila i ies be ween e al de elopmen and cance has long been ecognized, ollowing he disco e y o onco e al p o eins (such as α - e opo ein, ca cinoemb yonic an igen, human cho ionic gonado opic, o a e iew see [ 77 ]), which a e umo -associa ed an igens (TAAs) exp essed du ing emb yonic de elopmen and e-acqui ed in adul s du ing cance [ 78 ]. Human iPSCs sha e gene ic and ansc ip omic signa u es wi h cance issues, including se e al cance - ela ed genes and mo e han 100 human TAAs and umo -speci ic an igens (TSAs), which a e p o ein ma ke s ha can be ecognized by he immune sys em [79,80]. In con as o di e en ia ed cells, cance cells and ESCs e ain he abili y o e-en e he cell cycle and p oli e a e and i does no come o a su p ise ha common me abolic s a egies ha uel anabolic cell g ow h a e adop ed. Cance cells and ESCs p esen inc eased glycoly ic lux which sus ains high a es o cellula p oli e a ion by apidly gene a ing adenosine iphospha e (ATP), whe eas mi ochond ia is mainly used o anabolic p oduc ion, a phenomenon known as he Wa bu g e ec [ 81 – 83 ]. Howe e , ecen e idence sugges s ha ene ge ics o slow-cycling and he apy- esis an CSCs di e om he bulk p oli e a i e umo , ha la gely ely in glycolysis. CSCs p esen an inc eased dependency in OXPHOS and mi ochond ial espi a ion and his ulne abili y can be exploi ed o umo e adica ion [ 84 ]. Fo ESCs, he main me abolic p og am adop ed and ene ge ic uel used o ATP p oduc ion seems o ely on he plu ipo ency s a e [ 81 ]. When compa ed o p imed, nai e ESCs p esen ele a ed mi ochond ial unc ion, inc eased OXPHOS, and a lowe le el o glycolysis whe eas p imed ESCs p esen highe m DNA copy numbe and mo e ma u e mi ochond ia [81,82]. As o oncogenesis, cellula ep og amming o plu ipo ency induces a global me abolic emodeling ha leads o a p og essi e ansi ion om soma ic oxida i e me abolism o glycolysis, wi h an inc ease in glycoly ic a e and lac a e p oduc ion oge he wi h a dec ease in cellula espi a ion and o he mi ochond ial adap ions [ 85 , 86 ]. Me aboli es unc ion goes beyond se ing as subs a es o ene gy p oduc ion and anabolic g ow h and can de e mine he balance be ween CSCs and ESCs sel - enewal and di e en ia ion. Many me abolic in e media es (as ace yl-CoA, S-adenosylme hionine, α -ke oglu a a e) se e as obliga o y subs a es o co ac o s o ch oma in-modi ying enzymes, impac ing epigene ic egula ion and gene exp ession [ 82 ]. Fo ins ance, epide mal CSCs a oid epigene ically d i en di e en ia ion by shu ing down endogenous se ine syn hesis and becoming dependen on ex acellula se ine [ 87 ]. De no o se ine syn hesis s imula es α -ke oglu a a e-dependen dioxygenases ha emo e he ep essi e his one modi ica ion H3K27me3, ac i a ing di e en ia ion and blocking squamous cell ca cinoma g ow h [ 87 ]. Lipid me abolism is also linked o s em cell plu ipo ency. Fa y acid syn hesis is c i ical o ESCs plu ipo ency and cellula ep og amming in o iPSCs [ 88 ]. Likewise, s imula ion o de no o lipogenesis by lipid dep i a ion induces a nai e-like s a e in human PSCs by enhanced his one ace yla ion and dec eased DNA me hyla ion which mimics ERK inhibi ion [ 89 ]. These s udies highligh he po en ial o nu ien manipula ion as a s a egy o induce me abolic-d i en epigene ic and gene exp ession changes unde lying (CSCs and iPSCs) s em cell a e decisions. Fo no mal o CSCs o main ain hei s emness, hey mus balance bo h in insic and ex insic signals, main aining p oli e a ion while inhibi ing unwan ed di e en ia ion and bypassing immune ecogni ion. Epigene ic egula o s a e e ec o s o hese inpu s wi h al e a ions o en leading o cell dea h, o oncogenic ans o ma ion and g ow h [ 90 , 91 ]. Cance cells a e gene ically and epigene ically Cance s 2020,12, 3581 5 o 25 plas ic, ha ing he po en ial o egain benign cell unc ions ia e-exp ession o lineage-speci ic genes [ 90 , 92 ]. Recen ly iPSCs ha e been p oposed as a no el me hod o s udying ca cinogenesis h ough cance cell ep og amming. A numbe o human cance cell lines and p ima y samples ha e been used o gene a e iPSCs, in an a emp o ecapi ula e he mechanisms unde lying cance de elopmen and as a s a egy o induce ansi ion om malignancy o benigni y [ 6 , 90 , 93 ]. Since he ini ial desc ip ion o ep og amming using he ou Yamanaka TFs [ 29 , 30 ], se e al modi ica ions in he ep og amming p o ocol aiming a inc easing e iciency ha e been desc ibed. Many o hose, ac commonly in cance -associa ed pa hways and may con ibu e o umo p og ession, such as down- egula ion o he umo supp esso s p53 o p16/nk4a-ARF [ 94 , 95 ]. In line wi h his, we and o he s ha e p e iously demons a ed ha Zeb2, a ac o in ol ed in epi helial o mesenchymal ansi ion (EMT) and me as a ic po en ial [ 96 – 98 ], is an impo an ba ie limi ing he ep og amming o aged ib oblas s [ 99 ]. As o CSCs, iPSCs ha e he po en ial o in ini e p oli e a ion and in ce ain scena ios, umo igenic p ope ies ha e been a ibu ed. O no ice, all o he 4 TFs o iginally included in he Yamanaka cock ail ha e p o ed oncogenic po en ial [ 32 ], especially c-Myc which is one o he mos equen ly mu a ed genes in human cance s. Indeed, app oxima ely 20% o he chime ic mice o sp ing made by iPSCs-de i ed om 4TFs ep og amming de eloped umo s a ibu able o eac i a ion o he c-Myc ansgene [ 100 ]. Besides, ampli ica ion o genomic and m DNA mu a ions du ing he ex ensi e in i o iPSCs cul u ing may ep esen ano he sou ce o umo igenici y and exp ession o immunogenic p o eins [ 101 , 102 ]. Simila ly, ophec ode m ma ke s de ec ed in in i o iPSCs a e also linked o he pa hobiology o cance (e.g., Fg 2) [73]. CSCs ha e he abili y o bo h sel - enewal and di e en ia ion and CSCs su i e chemo he apy ha e been shown o be able o e-es ablish umo s [ 75 , 103 ]. While i s o igin is no ully known, i has been sugges ed ha CSCs could a ise as a esul o mu a ions in s em, ansien -ampli ying, di e en ia ed o cance cells leading o acquisi ion o malignan and/o s em cell p ope ies [ 92 ]. Mo eo e , a numbe o s udies also sugges he exis ence o plas ici y wi hin cance and s em cell’s compa men s [ 92 ]. Fo ins ance, in b eas cance , no mal and CSCs-like cells can a ise de no o om mo e di e en ia ed cell ypes e ealing ha mamma y s em cells encompass bidi ec ional in e con e sions be ween s em and non-s em compa men s [ 104 ]. A i icial (o in i o ) cellula ep og amming has also been widely compa ed o he biogenesis o CSCs [ 90 ]. T ansi o y induc ion o he 4TFs in mice is able o p oduce in i o ep og amming wi hin issues wi h o ma ion o e a omas and ci cula ing iPSCs in he blood [ 73 ]. Whe he ep og amming o soma ic di e en ia ed cells in o iPSCs is a (na u ally) occu ing p ocess in i o and can con ibu e o cance and s em cell popula ions emains o be elucida ed. 3. iPSCs as Whole-Cell Cance Vaccines Ou g owing knowledge o umo immunobiology, which is e lec ed by he cons an inc ease complexi y o he immunoedi ing concep has led, in he las decade, o a pa adigm shi in he ea men o nume ous cance s [ 105 – 107 ]. Blockade o he immune checkpoin s ep esen a ema kable b eak h ough in cance immuno he apy whe eas o he s a egies, such as TAAs/TSAs and DCs accines, s a o ise as alid con ibu es o push o wa d his h illing ield [ 108 ]. He e, au ologous whole umo cells accines seem o add ess he umo he e ogenei y ca ea . Howe e , his app oach equi es access o su icien iable biopsy ma e ial and can only be used in a he apeu ic se ing, wi hou possibili y o design p ophylac ic s a egies. As p e iously de ailed, cance ans o ma ion is coupled o he appea ance o s em-like ea u es, d i ing he idea ha s em/emb yonic issues may ha bo he same an igens p esen in umo s. In his con ex , ESCs and iPSCs ha e been ex ensi ely es ed in an i- umo immuniza ion p o ocols [ 79 , 109 , 110 ]. The seminal expe imen s o Geo ge Schöne demons a ing ha immuniza ion wi h emb yonic issue gene a es speci ic an i umo esponses da e back a cen u y ago [ 111 ]. Since hen, nume ous p e-clinical s udies ha e shown ha accina ion wi h emb yonal ma e ial p e en no only he g ow h o ansplan able umo s as well as umo igenesis caused by i al and chemical agen s ( e iewed in [ 112 ]). Ne e heless, e hical cons ains associa ed wi h he accessibili y o ESCs, he educed epe oi e o Cance s 2020,12, 3581 6 o 25 ma ching cance an igens and unwan ed immune esponses gene a ed by human leukocy e an igen (HLA) incompa ibili y in allogenic accine se ings, limi ed i s clinical applica ion [ 113 ]. In his ega d, au ologous iPSCs-based accines may add ess hese limi a ions p o iding an al e na i e s a egy. The i s epo o he use o iPSCs as a whole-cell cance accine comes om Li and collabo a o s, whe e hey compa e he e icacy o human iPSCs and ESCs lines as immunizing agen s in a ansplan able mouse colon ca cinoma model [ 114 ]. Al hough accina ion wi h bo h human ESCs and iPSC lines induced signi ican expansion o umo -speci ic IFN γ - and IL-4-p oducing T cells, only human ESC cells inhibi ed umo g ow h. The he e ogenei y o onco e al an igens exp ession as well as he accumula ion o myeloid-de i ed supp esso cells (MDSCs) in iPSCs-immunized g oups we e p oposed as possible explana ions o he dis inc an i- umo p o ec ion con e ed by iPSCs and ESCs [ 114 ] (Table 1). In e es ingly, no e idence o au oimmuni y was obse ed, sugges ing ha he an igens p esen in ESCs a e di e en om he an igens p esen ed in adul s em cell niches. One o he i s e idences ha au ologous iPSCs could elici an immunogenic esponse was p esen ed by Zhao e al., whe e ansplan a ion o imma u e C57BL/6 (B6)-de i ed iPSCs ( ep og ammed om mouse emb yonic ib oblas s) induced T cell in il a ion, issue damage and eg ession o e a omas in syngeneic ecipien mice [ 115 ] (Table 1). In his s udy, T cell in il a ion was obse ed in mos e a omas o med by he B6 iPSCs in syngeneic mice, some o which also exhibi issue nec osis and an appa en eg ession a 40 days pos -implan a ion [ 115 ], suppo ing ha iPSCs a e immunogenic in syngeneic ecipien s. The concep o iPSCs-based an icance accine was u he e ined by Koo eman by using i adia ed au ologous iPSCs in combina ion wi h he TLR9 agonis , CpG oligodeoxynucleo ide [ 80 ] (Table 1). The use o au ologous iPSCs allowed o minimize alloimmuni y s imula ed by MHC misma ches and he addi ion o he immunos imula o y adju an CpG p omo ed he ma u a ion o APCs such as DCs. In a p e en i e accina ion se ing, injec ion o i adia ed au ologous iPSCs plus CpG esul ed in he p oduc ion o umo - eac i e an ibodies, expansion o e ec o /memo y helpe T cells and ma u e DCs as well as in a signi ican dec ease in CD4 + CD25 + FoxP3 + egula o y T cells (T- egs). This a o able CD8 + T/T- egs a io in accina ed mice esul ed in he ejec ion o ansplan ed b eas cance , melanoma, and meso helioma umo cells [ 80 ]. Analysis o ea ly in a- umo in il a es e ealed inc eased numbe s o accine-elici ed B and T clones exp essing IL-2, IL-4, and IL-5. Mo eo e , adop i e ans e o T cells om immunized animals o naï e ecipien s con e ed umo p o ec ion, p o iding clea e idence ha he obse ed e ec was being media ed by T cells. When es ed in a clinically ele an scena io (i.e., he apeu ic adminis a ion in animals bea ing es ablished melanomas), he iPSCs +CpG accine ailed o educe umo g ow h. Recen ly, ano he s udy epo ed ha iPSCs he apy was ine ec i e in educing melanoma umo g ow h p obably due o he es ablished immunosupp essi e mic oen i onmen [ 116 ]. Howe e , as an adju an he apy a e melanoma esec ion, iPSCs accines eac i a ed he immune sys em in elimina ing aces o melanoma cells. This ejec ion was media ed h ough IL-4 exp essing B-cells, TNF- α -exp essing CD11b + GR1 hi myeloid cells, and a educ ion o umo -p omo ing Th17 cells [ 80 ]. This seminal obse a ion c ea ed a g ea en husiasm gi en ha i showed iPSCs as a TSAs- and TAAs sou ce, eadily a ailable a ime o umo esec ion, allowing o p ime and eac i a e an i umo immuni y and p e en ing elapses. Addi ional s udies co obo a ed he po en ial o iPSCs-based an icance accines in mul iple umo models. A accine composed o Hype -IL6 (H16) gene-modi ied melanoma cells admixed wi h allogenic iPSCs was shown o cause supe io ex ension disease- ee su i al, and long- e m o e all su i al in melanoma-challenged C57BL/6 mice when compa ed o modi ied cells alone o H16 modi ied cells plus ESCs [ 117 ] (Table 1). Mice accina ed wi h mixed-iPSCs accine shown, in he injec ion si e and spleen, inc eased numbe s o in lamma o y monocy es, ac i a ed CD4 + T cells and memo y CD4 + T and CD8 + T cells, while he e was a dec ease in he pe cen age o MDSCs. iPSCs immuniza ion also led o inc eased pe cen ages o in lamma o y monocy es, DCs and NKs in he umo mic oen i onmen (TME) as well as supe io le els o IFN- γ and IL-12p70, causing concomi an ly a educ ion o in il a ing T egs [ 117 ]. Mo eo e , Gabka-Buszek and colleagues demons a ed he Cance s 2020,12, 3581 7 o 25 e ec i eness o melanoma accines composed o umo cells mixed wi h melanoma s em-like cells o iPSCs. In pa icula , a accine con aining syngeneic iPSCs and B16F10 cells, signi ican ly inhibi ed umo g ow h and inc eased mouse su i al [117]. Addi ionally, in a humanized-mice model o lung cance , accina ion wi h iPSCs +CpG was shown o inc ease he pe cen age o splenic APCs and cy o oxic T cells, ci cula ing e ec o /memo y CD4 + and CD8 + T cells and umo -in il a ing CD8 + T cells, while dec easing T egs [ 118 ] (Table 1). This immune landscape suppo ed an e ec i e supp ession o umo g ow h, and he cen al ole o umo an igen-speci ic T cells was demons a ed by he p o ec ion con e ed h ough he adop i e ans e o spleen T cells om he accine p eimmunized mice o un accina ed ecipien s. The p o ec i e immuni y elici ed by iPSCs was p oposed o be due o he gene exp ession pa e n sha ed be ween iPSCs and lung adenoca cinoma s em cells [118]. Cance s 2020,12, 3581 8 o 25 Table 1. Immunogenici y esponses elici ed by induced plu ipo en s em cells (iPSCs) whole-cell cance accines in mouse models. Species Cell o O igin Rep og amming Condi ions iPSCs S a e Local o Eng a men Immunogenici y Tes Immune Response Immunogenic P o eins Tumo Ta ge ed S udy Homo sapiens Fe al lung ib oblas line IMR90 Len i i us exp essing 6F 1TTZ1 cell line sc 2Te a omas Tumo -speci ic IFN-γ-and IL-4-p oducing T cells Onco e al an igens Colon ca cinoma [114] Mus Musculus MEFs 3 Re o i us exp essing 3F 4 and 4F 5, 4F non-in eg a i e episomal ec o SSEA-1 sc (hind leg) Te a omas IFN-γ eleasing assay in i o CD4+helpe T CD8+ cy o oxic T Ho mad1 Zg16 na 6[115] Mus Musculus Fib oblas s om FVB, C57BL/6J, and CBA/J mice Codon-op imized mini-in onic plasmid con aining 4F SSEA-1 sc ( lank) Te a omas Inc ease in e ec o /memo y helpe T cells, ma u e DCs, IL-4-exp essing B cells, TNF-αexp essing myeloid cells; dec ease in T egs and Th17 cells Onco e al an igens B eas cance , melanoma, and meso helioma umo cells [80] Mus Musculus MEFs Nucleo ec ion wi h plasmid coding 4F SSEA-1; Epcam, E-cadhe in, NANOG, alkaline phospha ase sc na In lamma o y monocy es, ac i a ed CD4+T cells, memo y CD4 + T and CD8 + T; umo in il a ing DCs, NKs, in lamma o y monocy es; dec ease in T egs and MDSCs TAAs 7/TSAs 8sha ed wi h CSCs 9 Melanoma [117] Homo sapiens Fib oblas s na na sc na Splenic APCs and cy o oxic T cells; ci cula ing e ec o /memo y CD4+and CD8+T cells; umo in il a ing CD8+T cells; dec ease in T egs TAAs/TSAs sha ed wi h CSCs Lung cance [118] Mus Musculus Tail- ip ib oblas s Re o i us exp essing 3F Endo ac o s; colony o ma ion assays sc IFN-γ eleasing assay in i o Inc ease in ac i a ed CD8+ and CD4+T cells TAAs/TSAs Panc ea ic duc al adenoca cinoma [119] 1 6F (Oc 3/4/Sox2/Lin28/Kl 4/Nanog/c-Myc); 2 subcu aneous; 3 mouse emb yonic ib oblas s; 4 3F (Oc 4/Sox2/Kl 4); 5 4F (Oc 4/Sox2/Kl 4/cMyc); 6 non-add essed; 7 umo associa ed an igens; 8 umo speci ic an igens; 9cance s em cells. Cance s 2020,12, 3581 9 o 25 Al hough he di e en ia ion o iPSCs is epo ed o esul in loss o immunogenici y [ 80 , 120 , 121 ], iPSCs-de i ed panc ea ic umo cells we e ecen ly p oposed as possible whole cell- accines o p e en o delay panc ea ic duc al adenoca cinoma (PDAC) in KPC ansgenic mice [ 119 ] (Table 1). Tumo cells we e c ea ed by in si u gene edi ing o heal hy ib oblas -de i ed iPSCs ia in oduc ion o K as and p53 mu a ions. In con as o non ans o med iPSCs, he ansc ip ome o ep og amed cells e ealed highly simila gene exp ession p o iles o ansgenic mouse-de i ed umo cell lines, alida ing he model as a sou ce o TAAs and TSAs. To inc ease he immunogenici y, iPSC-de i ed panc ea ic umo cells we e in ec ed wi h adeno i us o accinia i us p io inocula ion. The p ophylac ic accina ion o KPC ansgenic mice wi h adeno i us-in ec ed ep og amed iPSCs ollowed by boos ing wi h he accinia-in ec ed cells was shown o e ec i ely delay PDAC de elopmen , signi ican ly p olonging mice su i al. Tumo con ol was associa ed wi h inc eased le els o ac i a ed CD8 + and CD4 + T ound in lymph nodes, spleen, and umo in il a es. Tumo speci ic immuni y was howe e los o e ime and in il a ion o CD8 + T cells wi hin umo s was minimal 3 mon hs a e immuniza ion [ 119 ]. O e all, hese s udies sugges ha au ologous iPSCs elici a speci ic an i- umo al esponse, highligh ing he po en ial use o iPSCs-based accines o cance he apy. 3.1. Lessons F om ESCs: The Immune Response o Plu ipo en Cells Pionee s udies using ESCs o cance accina ion shown e idence o p o ec i e e ec s only when ea ly bu no la e emb yonic issues we e used and his was a ibu ed o he exp ession o onco e al an igens [ 77 , 112 , 114 ]. Al hough some s udies ha e used ESCs in an allogeneic ans e , due o he limi ed a ailabili y, ESCs cells a e mo e likely o be ans e ed o an un ela ed hos which may igge an alloimmune esponse. Thus, al hough indis inguishable om allogeneic esponses, an immune esponse o onco e al an igens is gene a ed when ESCs a e ans e ed ei he allogeneically o syngeneically. The immunogenic esponse elici ed by iPSCs was sugges ed o con as wi h ESCs, whe e ansplan a ion in o syngeneic ecipien mice esul ed in e icien o ma ion o e a omas wi hou e idence o immune ejec ion (as indica ed by he lack o de ec able CD4 + T cell in il a ion) [ 115 ]. ESCs possess immune p i ileged p ope ies and ha e he capaci y o inhibi immune ac i a ion. Howe e , he immunogenici y o ESCs may ha e been unde es ima ed [ 122 ]. Undi e en ia ed s em cells and de i a i es ha e been shown o induce an immune esponse in i o ha in ol es cy o oxic T lymphocy es, helpe T cells, and NKs [ 123 – 125 ]. Indeed, ESCs exp ess NK cell–ac i a ing ligands and a e suscep ible o NK cell ecogni ion and a ack [ 126 , 127 ] al hough some s udies claim ha undi e en ia ed ES cells a e esis an o NK cell a ack [128–130]. The e a e wo main classes o polymo phic MHC molecules, human leukocy e an igen (HLA) in humans, exp essed by cells ha a e able o p esen an igens [ 131 ]. While mouse ESCs exp ess mRNA o MHC molecules, bu no he co esponding p o eins [ 132 – 134 ], human ESCs exp ess a iable, albei low le els o HLA class I molecules and almos unde ec able le els o HLA class II [ 125 , 129 , 132 , 133 , 135 – 137 ]. As undi e en ia ed ESCs exp ess low le els o MHC-I and co-s imula o y molecules, ansplan ed g a -de i ed an igens may be p ocessed di ec ly by APCs o indi ec ly h ough APCs ac i a ion o T cells [ 129 , 138 , 139 ]. In his con ex , he immune esponse o ansplan ed ESCs may in ol e 3 main de elopmen al s ages. The i s s age occu s du ing which ecipien MHC class II– es ic ed CD4 + T cells ecognize an igens p esen ed by ecipien APCs and elease p oin lamma o y cy okines. The second s age occu s when sel - es ic ed CD4 + T cells help o gene a e cy o oxic T lymphocy es ha can ecognize in ac MHC class I molecules. The hi d s age is an ibody esponse, du ing which alloan igen-p imed CD4+T cells deli e ac i a ing signals o B cells. The low le els o MHC an igens exp essed by ESCs a e belie ed o con ibu e o T cell ecogni ion a oidance e en hough in i o ESCs could ins ead be ecognized and suscep ible o killing by NKs [ 126 , 132 , 136 ]. Independen ly o he immunologic condi ion o ESCs ans e (syngeneic, allogeneic and xenogeneic), he inna e immune esponse in ol ing NKs plays a c i ical ole agains ansplan ed ESCs. Mo eo e , MHC exp ession can be inc eased du ing ESCs di e en ia ion and in he p esence Cance s 2020,12, 3581 16 o 25 Acknowledgmen s: We since ely apologize o colleagues whose wo k has no been included in his Re iew due o space limi a ions. Con lic s o In e es : The au ho s decla e no con lic o in e es . Re e ences 1. 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