Embryonic stem cells: a novel source of dendritic cells for clinical applications
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International Immunopharmacology 5 (2005) 13–21 www.elsevier.com/locate/intimp
Embryonic stem cells: a novel source of dendritic cells for clinical applications Paul J. Fairchild*, Kathleen F. Nolan, Siaˆ n Cartland, Herman Waldmann The University of Oxford, Sir William Dunn School of Pathology, South Parks Road, Oxford, OX1 3RE, UK
Abstract
As arbit arbitrato rators rs of the immune response, response, dendritic dendritic cells (DC) are uniquely uniquely plac placed ed to nego negotiate tiate the balan balance ce betw between een the opposing forces of tolerance and immunity, making them attractive candidates for clinical applications. Accordingly, DC have been used successfully in the treatment of cancer, enhancing immune responses to tumour-asso tumour-associated ciated antigens (TA (TAA) A) in experimental animal models and phase I clinical trials. A novel source of DC that has recently been described is the embryonic stem (ES) cell whose differentiati differentiation on in vitro may be dire directed cted along multiple lineage pathways. pathways. Such pluri pluripote potency ncy offers unparalleled opportunities for the treatment of chronic and degenerative disease states by the replacement of affected tissues, a vision which has inspired the emerging field of regenerative medicine. By sharing the genotype of therapeutic cell types, such as cardiomy card iomyocyt ocytes es and dopaminergi dopaminergicc neur neurons ons derived from the same ES cell line, so-c so-called alled esDC may offer pros prospects pects for reprogramming the immune system to tolerate the grafted tissues. Here, we describe how the unique properties of esDC and the ES cells from which they derive, make them eminently suited to clinical applications, overcoming many of the issues that currently limit the effectiveness of DC-based immune intervention. D 2004 Elsevier B.V. All rights reserved. Keywords: Keywor ds: Dendritic cells; Embryonic stem cells; Regenerative medicine; Tolerance
1. Introduction
Recent dec Recent decad ades es hav havee wi witne tness ssed ed a pr progr ogres essi sive ve chan ch ange ge in em emph phas asis is in th thee he heal alth thca care re ne need edss of develope deve loped d coun countrie tries. s. Due part partly ly to impr improved oved livi living ng conditionss and signif condition significant icant advances in the treatment of infectious microorganisms, life expectancy in Western soci so cieti eties es ha hass ri rise sen, n, un unaba abated ted,, si sinc ncee the 184 1840s 0s and * Corres Correspondin ponding g author. Tel.: Tel.: +44 1865 275606; fax: +44 1865 275501. E-mail addr address: ess: [email protected] (P.J. Fairchild). D
1567-5769/$ - see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2004.09.005
shows no indication of having reached a plateau [1] 1].. While increased longevity is undoubtedly one of the succes suc cesses ses of mod modern ern medicine, medicine, it has been acc accomom panied by a dramatic increase in incidence of chronic and an d de dege gener nerati ative ve dis disea ease se st stat ates es wh which ich thr threat eaten en to dominate healthcare resources in the future [ [2] 2].. Since many man y con conditi ditions, ons, such as diab diabetes etes and Par Parkins kinson’ on’ss disease, may be amenable to cell replacement therapy (CRT), (CR T), the adve advent nt of plur pluripot ipotent ent hum human an emb embryon ryonic ic stem (ES) cells cells [3] [3] has has received considerable acclaim by promi promising sing to meet the grow growing ing deman demand d for replacement cell types and tissues [4] tissues [4].. Indeed, current
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estimates tes suggest that upwards of 3000 Americ Americans ans die estima each day of conditions that may ultimately be treated trea ted through thr ough the pana panacea cea of reg regener enerativ ativee med medicin icinee [5] [5].. Altho Al though ugh va vari rious ous ani anima mall mo mode dels ls of dis diseas easee ha have ve proven prove n amenable to treatment by such an approach
phenotype and propensity for long-term storage. While CD34+ stem cells may be stored frozen over prolonged periods of time, access to such progenitors is largely constra con strained ined by the lim limited ited ava availab ilabilit ility y of umb umbilic ilical al cor cord d blood; conversely, DC differentiated from the patient’s
[6–8], translation to the clinic will require an effective [6–8], strategy for addressing the immunological barriers of histoincompatib histoin compatibility ility between the recipient of CRT and the ES cell line from which the tissues are derived. A promising approach to this problem lies in the dual du al ca capa paci city ty of ES ce cell llss to ac actt as a so sour urce ce of therapeutic cell types to ameliorate the progression of dise di seas asee an and d of le leuk ukoc ocyt ytee su subs bset etss to en ensu sure re th thei eir r acceptancee th acceptanc throu rough gh the indu inductio ction n of tra transpl nsplant antatio ation n tolerance [9] [9].. Given Given the eme emergi rging ng rol rolee of den dendrit dritic ic cells (DC) in the maintenance of self-tolerance under steady state conditions and their ability to reestablish tolerance tolera nce in both both auto autoimm immune une and allo alloimm immune une set set-tings [10–12], [10–12], the description of of protocols protocols for their
own per periph iphera erall bloo blood d mon monocyt ocytes es are mor moree rea readily dily available but may show less phenotypic stability with time, displaying an inherent tendency for spontaneous maturation in culture. Secondly, an issue which has proven to be the rate-limiting step in cancer immunotherapy has been been the identificat identification ion of appropriate appropriate target antigens and the most effective form for their delivery to DC; syn synthe thetic tic pep peptid tides, es, re recom combin binant ant TAA and unfrac unf raction tionated ated tumo tumour ur lys lysates ates have each been use used d with varying success. While endogenous expression of TAA is wi widel dely y be belie lieved ved to be th thee mo most st ap appr propr opria iate te fo form rm of delivery by virtue of the access it provides to both MHC class I and II pathways of antigen antigen processing, processing, the the approach is limited by the need for effective protocols
derivation from mouse ES cells [13,14] cells [13,14] represents represents an important step towards realising this goal. While Wh ile th thee tol toler erog ogeni enici city ty of DC ha hass be been en ap appr prec eciat iated ed forr so fo some me ye year ars, s, an un under dersta standi nding ng of th thee mo mole lecul cular ar ba basi siss of this phenomenon remains less well defined than the mechanis mec hanisms ms that unde underlie rlie imm immunoge unogenici nicity ty.. Cons Conseequently, the use of DC in a therapeutic context has so far been largely restricted to strategies for vaccination to tumour tumour-assoc -associated iated antigen antigenss (T (TAA) AA) [15]. [15]. Such an approach to cancer immunotherapy has enjoyed considerable sidera ble success in animal models of diseas diseasee [16–18] and has inspired a number of small-scale clinical trials for the treatment of melanoma, lymphoma, myeloma, as wel welll as pro prost stat atee an and d re rena nall ca cance ncer r [19,20]. [19,20]. The expansion of tumour-specific cytotoxic T lymphocytes (CTL) in these studies has correlated correlated with regression regression of establ est ablishe ished d tum tumours ours and sec second ondary ary met metasta astases ses in a proportion of patients, although alth ough clinical outcomes have varied considerably between trials trials.. This inconsistency has highlighted the need to optimise and standardise protocols for the derivation, deriv ation, culture and an d administration administ ration of DC; indeed, in the 8 years since the first trial was performed, various issues have been identified that must be addressed before DC-based treatment regimes may be offer offered ed as reliab reliable le altern alternatives atives to conventio conventional nal therapies [21,22] therapies [21,22].. Firstly, identification of the most appropriate source of DC remains to be resolved, resolved, the main criter criteria ia for their use in vivo being their availability, yield, stability of
for genetic modification of the DC inoculum, which remains a significant obstacle. Although the development of retroviral and adenoviral vectors has begun to fulf fu lfill ill th this is ne need ed,, th thei eirr us usee in a cl clini inica call co conte ntext xt introduces a significant element of risk ris k which may be difficult to justify on ethical grounds [ grounds [23] 23].. Thesee cons Thes consider ideratio ations ns hel help p illu illustra strate te the pre pressi ssing ng need for quality control of preparations of DC intended for clinical use, so as to ensure both safety and efficacy of the pr proce ocedur dure. e. Cl Clea early rly,, an any y so sour urce ce of DC tha thatt pe perm rmits its consist con sistency ency and rep reprodu roducibi cibility lity betw between een bat batches ches,, whil whilee providing providin g ample opportu opportunities nities for quality control, would wou ld off offer er sig signif nifican icantt adva advantag ntages es ove overr the curr current ent art. ar t. Alt Althou hough gh ef effo fort rtss are pr pres esent ently ly un under derway way in a number of laboratories to secure the differentiation of DC from human ES cells, we discuss below how our own experiences of esDC in the mouse suggest that they fulfil many of these requirements, auguring well for their ultimate application to the clinic.
2. Materials and methods
2.1. ES cell lines and mice
ESF116 is an ES cell line derived from CBA/Ca mice mi ce wh whic ich h is ka kary ryot otyp ypic ical ally ly ma male le an and d ha hass be been en described in detail previously [ previously [13] 13].. ESF121 is likewise of CBA/Ca origin but is karyotypically female. Both
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lines display germline competence competence and were cultured according to standard protocols [14] [14].. C57Bl/10 mice were we re us used ed as a so sour urce ce of al allo loge gene neic ic T ce cell lls. s. / A1xRAG1 micee tra mic transg nsgenic enically ally exp expres resss a T cell receptor (TCR) (TCR) specific for the 479–493 epitope of the
dry ic dry icee be befo fore re bei being ng st store ored d lon long g ter term m un under der li liqui quid d nitrogen. To induce the maturation of esDC, replated cells were exposed to 1 Ag/ml of lipopolysaccharide (LPS) from E. coli Serotype 0127:B8 (Sigma). After over-
male specific antigen, Dby, k in the context of the MHC clas cl asss II det deter ermi minan nant, t, H2 H2E E . Fe Fema male le A1 A1xRA xRAG1 G1/ mice mi ce we were, re, theref therefor ore, e, us used ed as a so sour urce ce of T cel cells ls specificc for this well-def specifi well-defined ined minor histocom histocompatibilit patibility y (mH) antigen.
night culture, the medium was replaced and the cells cult cu ltur ured ed fo forr a fu furt rthe herr 24 h: ma matu ture re es esDC DC we were re harve har vest sted ed the fo follo llowi wing ng da day y as the non nonad adhe here rent nt populati popu lation. on. Popul Population ationss of contr control ol bone marrow derived DC (bmDC) were obtained by culturing bone marrow cells from CBA/Ca mice in medium supplemented with 25 ng/ml of GM-CSF. Cultures were fed by replacement of the medium on days 3 and 6 of culture cult ure and matu maturat ration ion was spe specifi cificall cally y ind induced uced by addition of 1 Ag/ml of LPS on day 6.
2.2. Directed differentiation of ES cells
Protocols for directing the differentiation of pluri potent ES cells towards the DC linea ineage ge have been described in detail elsewhere [14]. [14]. Briefly, ES cells weree cult wer culture ured d on mito mitotica tically lly inac inactiva tivated ted emb embryon ryonic ic fibr fi brobl oblas astt fe feed eder er ce cell llss in DM DMEM EM (G (Gibc ibco) o) su supp pplelemented with 15% FCS, 2 mM l-gl -glutam utamine ine,, 1 mM 5
sodium pyruvate and 510 M 2-mercaptoethanol. Genetic modification of the parent ES cell ce ll lines lines was achie ac hieved ved by lip lipof ofec ectio tion n as de desc scrib ribed ed [14]. [14]. Stable transf tra nsfecta ectants nts were sele selected cted by neom neomycin ycin res resista istance nce and were cloned at the single-cell level to produce individual ES cell lines which were stored frozen. As a prelude to their differentiation, ES cells were passag pas saged ed twi twice ce in gel gelati atinis nised ed fla flasks sks in med medium ium supplemented with 1000 U/ml of recombinant leukaemia inhibitory factor (rLIF; Chemicon) to remove the feeder cells. ES cells were then cultured in suspension at lo low w de dens nsit ity y (410 5 ce cell llss pe perr 90 90-m -mm m di dish sh of bacteriological plastic) so as to permit the formation of em embry bryoid oid bod bodie iess (E (EB) B).. Af After ter 14 day days, s, EB wer weree plated onto tissue t issue culture cult ure plastic in medium medi um containing containi ng 200 U/ml rec recombi ombinant nant inte interleu rleukinkin-3 3 (IL(IL-3; 3; R&D Systems Sys tems)) and 25 ng/ ng/ml ml of gra granulo nulocyte cyte-ma -macro crophag phagee colony colo ny sti stimula mulating ting fac factor tor (GM (GM-CS -CSF) F) to sup support port the outgrowth of DC. ES ce cell ll-d -der eriv ived ed DC we were re ha harv rves este ted d by ge gent ntle le pipetting to release lightly adherent cells, and were replated in fresh medium containing IL-3 and GMCSF CS F, to pe perm rmit it the ad adhe here renc ncee of co cont ntam amina inatin ting g stro st roma mall ce cells lls.. Af Afte terr ove overn rnigh ightt cu cultu lture re,, es esDC DC wer weree agai ag ain n di disl slod odge ged d by ge gent ntle le pi pipe pett ttin ing g an and d us used ed in func fu nctio tiona nall as assa says ys or st stor ored ed fr froze ozen n fo forr fu futur turee us use. e. Cryopre Cryo preser servati vation on was achi achieved eved by res resuspe uspendin nding g 6 esDC es DC at a de dens nsit ity y of 510 cel cells/ ls/ml ml in med medium ium containing 10% DMSO. Cells were frozen rapidly on
2.3. Proliferative assays
Mature Matu re or imm immatu ature re esD esDC C (5104 ce cell llss pe perr we well ll of a 96-well round-bottomed plate) were cocultured with 2105 nylon wool purified T cells from either allogeneicc C57B nei C57Bl/10 l/10 or TCR tra transge nsgenic nic A1xR A1xRAG1 AG1/ female mice. mic e. Aft After er 2 day dayss of cult culture ure,, prol prolife iferati ration on was ass assess essed ed 3 by pulsing with 25 ACi/well of H-thymidine deoxyribose (TdR; Amersham). Cultures were harvested and 3 H-TdR H-T dR inc incorp orpora oratio tion n me measu asure red d usi using ng a fla flatt bed scintillation counter. 2.4. Flow cytometry
The surface phenotype of DC was assessed by flow cytome cyt ometry try usi using ng FIT FITC-c C-conju onjugat gated ed mon monoclo oclonal nal anti anti- bodies (mAb) to MHC class II (Clone OX6; mouse IgG1) IgG 1) or biot biotinyl inylated ated anti anti-CD -CD86 86 (Cl (Clone one GLGL-1; 1; Rat IgG2a), followed by ExtrAvidin-FITC (Sigma). Irrelevant, species- and isotype-matched mAb were used as negative controls. Cells were stained on ice for 45 min, washed and fixed in 1% formaldehyde before being analysed by means of a Becton Dickinson FACSCal ACSCalibur ibur using CellQue CellQuest st softwa software. re.
3. Results
3.1. Yield and storage of esDC
Protoc Prot ocol olss de deve velo lope ped d in ou ourr la labo bora rato tory ry fo forr th thee differentiation of DC from mouse ES cell lines make
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icroenviron vironment ment conduc conducive ive to use of EB as a m icroen hematopoiesis [13,14] hematopoiesis [13,14].. EB are macroscopic structures derived from the sustained proliferation of single ES cell ce llss ma maint intai ained ned in su susp spens ension ion cu cultu lture re and mi mimi micc deve de velop lopme ment nt of th thee ea earl rly y em embry bryo o in all but their propensity for pattern formation. Despite these limitation tat ions, s, EB fre freque quentl ntly y dev develo elop p cys cystic tic str struct ucture uress analogous to the visceral yolk sac, the most primitive of all hematopoietic tissues. When plated onto tissue culture plastic, to which they may adhere, EB support the outg outgrowt rowth h of colo colonies nies cont containi aining ng der derivat ivatives ives of each ea ch of th thee th thre reee em embr bryo yoni nicc ge germ rm la laye yers rs in an inherently chaotic manner. The addition of GM-CSF and IL-3 to cultures steers mesodermal differentiation overwhelmingly towards the myeloid lineage, favouring the app appear earance ance of alm almost ost lim limitle itless ss numb numbers ers of terminally differentiated DC. We estimate that a single EB is capable of sustaining the development of more than 2107 esDC, which, once established in culture, may be harvested periodically every 4–5 days before eventua eve ntually lly succ succumb umbing ing to repl replica icative tive sen senesc escence ence.. Such Su ch a deg degre reee of exp expan ansio sion n is unr unriva ivalle lled d am among ong conv co nven enti tion onal al so sour urce cess of DC DC,, en ensu suri ring ng the re read ady y availability of material for clinical applications. Since a single passage of the parent ES cell line has the potential to yield several million EB, each with the capacity to spawn over 10 7 esDC, there is a significant likelihood that cells may be generated that are surplus to requ requirem irements. ents. We have have,, ther therefor efore, e, inves investigat tigated ed whether esDC may be cryopreserved for subsequent use.. Sign use Signific ificantl antly y, sna snap-fr p-freez eezing ing of cel cells ls in med medium ium supplemented with 10% DMSO permits their storage under liquid nitrogen for protracted periods of time with wi th no de detec tecta table ble los losss of via viabil bility ity.. Fu Furt rther hermo more re,, esDC stored in this way show no loss of functional integrit inte grity y, resp respondin onding g appr appropri opriatel ately y to matu maturati ration on stimulii and performing identically to fresh stimul freshly ly harvest harvested ed cell ce llss as st stim imul ulat ator orss of na R ve, v e, al allo loge gene neic ic T ce cell ll responses (Fig. (Fig. 1) 1). 3.2. Phenotypic stability of esDC in culture
A feature of esDC which distinguishes them from DC derived from bone marrow progenitors (bmDC), is the stability of their phenotype and function over time. Fig. 2 shows how bmDC are prone to spontaneous ne ous ma matur turat ation ion in cu cultu lture re,, as ev evide idence nced d by th thee progressive translocation of MHC class II to the cell
Fig. 1. Immunogenicity of esDC cryopreserved for 2 weeks in liquid nitrogen. Embryonic stem-cell-derived DCs were frozen at 10 7/ml in medium supplemented with 10% DMSO. Upon thawing, cells were plated overnight and induced to mature by addition of 1 A g/ml of LPS before being used as stimulators of na R ve, ve, allogeneic T cells (.). Freshly harvested mature ( w ) and immature esDC (n) were included for comparison.
surface (Fig. (Fig. 2A–C) 2A–C) and up-regulation up-regulation of of costimulatory molecules, such as CD86 (Fig. ( Fig. 2E–G), 2E–G), even in the absence of exogenously added stimuli. In contrast, esDC remain unchanged phenotypically during many weeks in culture, appearing largely indistinguishable from fr om th thee mo most st im imma matu ture re bm bmDC DC af afte terr 35 da days ys in cultur cul turee (Fi Fig. g. 2D an and d H) H).. Su Such ch ob obse serv rvat atio ions ns ar aree relevan rel evantt in the light of rec recent ent stu studies dies showing showing tha that t admini adm inistr strati ation on to hea health lthy y vol volunt unteer eerss of im immat mature ure monocy mon ocytete-der derive ived d DC pul pulsed sed wit with h pep peptid tides es fr from om influenza virus matrix protein (MP) suppresses MPspecific CD8+ CTL through the generation of IL-10secreting regulatory T cells [24] 24].. These observations are consistent with studies in the mouse suggesting thatt anti tha antigen gen pre present sentatio ation n by imm immatur aturee DC pola polarise risess respond res ponding ing T cell cellss towa towards rds a reg regulat ulatory ory phe phenoty notype pe and may contribute to the maintenance of peripheral tolerance [25,26] tolerance [25,26].. The arrest of esDC at an immature stage of the DC life cycle may, therefore, favour a protolerogenic phenotype, endorsing plans for their use as a conditioning regimen for CRT. 3.3. Antigen processing and presentation
The need to identify appropriate antigens against which wh ich to tar targe gett th thee im immu mune ne re respo spons nsee ha hass gr grea eatly tly limited DC-based strategies for cancer immunotherimmunother-
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Fig. 2. Comparison of phenotypic stability among esDC and DC derived from bone marrow progenitors. Histograms represent the levels of expression of MHC class II (A–D) and CD86 (E–H) by bmDC at day 9 (A and E) and day 14 of culture (B and F), showing spontaneous maturation of a significant proportion of cells over time. For comparison, (C) and (G) depict expression levels of the respective surface markers by bmDC, induced to mature in response to overnight incubation with LPS. (D) and (H) represent levels of surface expression by esDC maintained continuously in culture for 35 days, showing a similar phenotypic profile to the most immature populations of bmDC (A and E).
apy. Despite considerable efforts over the past decade to ide ident ntif ify y TAA, the nu numb mber er of pr prot otei eins ns tr truly uly restricted in their distribution to transformed cells is surp su rpri risin singl gly y sm smal all, l, ev even en fo forr me mela lanom noma, a, the be best st defined of all tumours at the biochemical level. And yet, targeting destructive immune responses to antigens ge ns tha thatt ar aree mo more re wid widel ely y di diss ssem emina inated ted tha than n the tumour tum our itse itself lf may hav havee pote potentia ntially lly dele deleter terious ious consequences. Mice engineered to express a neoantigen in pancre pancreatic atic h cells that was shared by a tumour cell line with which they had been inoculated, developed fatal autoimmune responses responses when vaccinated with DC presenting presenti ng the same antige antigen n in an immunog immunogenic enic fashion [27] [27].. Although DC have been generally well tolerated during clinical trials, autoimmune sequalae in th thee fo form rm of vi viti tili ligo go ha have ve be been en rep epor orte ted d as a consequence of vaccination against TAA expressed by melan me lanom omaa but sha share red d by nor normal mal,, unt untra ransf nsform ormed ed melanocytes, illustrating the need for caution [21] [21].. Importantly, the issues of antigen identity and the form and vehicle for its delivery to DC are rendered irrelevant when exploiting esDC for the induction of transplantation tolerance to tissues derived from the
same ES ce same cell ll li line ne.. Ha Havi ving ng di diff ffer eren enti tiat ated ed fr from om a common progenitor, esDC naturally share all relevant major and minor histocompatibility antigens with the therap the rapeuti euticc cel celll type types, s, thei theirr endo endogeno genous us exp expres ression sion obviating the need for the provision of antigen in an appr ap prop opri riat atee fo form rm fo forr up upta take ke by DC DC.. In Inde deed ed,, th thee molec mo lecula ularr ide identi ntity ty of the tra trans nspla planta ntatio tion n ant antige igens ns may become little more than academic academic rat rather her than representing an insurmountable obstacle to the implementati men tation on of imm immunot unother herapy apy.. The iss issues ues of MHC restriction that greatly complicate the use of peptide vaccines are also circumvented, since esDC may be entru en truste sted d wit with h the tas task k of se selec lectin ting g app approp ropria riate te epitope epi topess for presentati presentation on to the T cell rep repert ertoire oire in thee co th cont ntex extt of bo both th MH MHC C cl clas asss I an and d cl clas asss II determinants. Furthermore, the risk of eliciting erroneous autoimmune autoimmune responses is minimis minimised ed by the lack of all alloan oantig tigen en exp expres ressio sion n by the rec recipi ipient ent’’s own somatic tissues and the tolerogenic context in which any shared autoantigenic epitopes are presented. In or orde derr to inv inves estig tigat atee th thee ca capac pacity ity of esD esDC C to present endogenously expressed mH antigens, we have made use of A1xRAG1 / mice, transg transgenically enically
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expressing a TCR specific for the immunodominant 479–493 epitope of the male-restricted antigen, Dby, in the context of H2Ek . Na R ve, ve, splenic T cells from / femalee A1xRAG1 femal mice responded vigorously to mature mat ure esDC diff differe erentiat ntiated ed fro from m a kary karyotyp otypica ically lly
welcomee opp welcom oppor ortun tuniti ities es to su subtl btly y int inter erven venee in the outcome out come of the imm immune une res respons ponse. e. Nev Neverth ertheles eless, s, all sources of DC, described to date, display significant barriers to the introduction of heterologous genes and constructs. While lipofection is entirel is entirely y inadequate as
male ES cell line of CBA/ Ca C a origi origin, n, bu butt fa faile iled d to resp re spon ond d to fe fema male le es esDC DC (Fi Fig. g. 3A). Neverth Nevertheless, eless, addit ad ditio ion n of the sy synth nthet etic ic Db Dby y pe pepti ptide de to cultures cultures restored maximal responses in either case (Fig. (Fig. 3B). 3B). Thes Th esee re resu sults lts de demo monst nstra rate te th thee abi abilit lity y of es esDC DC to process an endogenously expressed mH antigen and present the immunodominant epitopes in the context of MHC class II determinants, a necessary requirement for the induction of transplantation tolerance.
The advantages that routine genetic modification of DC would offer to strategies of immune intervention
an app appro roach ach to tr tran ansf sfect ectio ion n (Fi Fig. g. 4A) A),, us usee of th thee cationic peptide, CL22, as a vehicle for delivery of plasmid DNA has enjoyed some measure of success, up to 17 17% % of mo mous usee DC ha havi ving ng be been en in indu duce ced d to express expr ess the enha enhanced nced gr een e en flu fluores orescent cent prot protein ein (EGFP)) reporte (EGFP reporterr gene [28] 28].. The The us usee of mR mRNA NA fo for r the purpose of tr ansf ansf ection ection has also proven surprisingly ingl y eff effecti ective ve [29], [29], althou although gh exp expre ress ssion ion of the transcribed gene is necessarily transient and has yet to exceed a transduction efficiency of 20%. Both of these approaches, while adequate to secure priming to TAA AA,, ar aree fa farr to too o in inef effi fici cien entt as a st stra rate tegy gy fo for r modifyi mod ifying ng cel cellula lularr phen phenotyp otypee and fun functio ction. n. Ind Indeed eed,, the only approach to genetic modification that comes
extend far beyond the expression of appropr appropriate iate TAA. Thee abi Th abilit lity y to ove overe rexpr xpres esss or fu func nctio tional nally ly si sile lence nce genes involved in immune regulation would provide
close to such a remit is the use of viral vectors, such as adenovirus, through which the stable introduction of transgenes may be achieved in up to 80% of cells.
3.4. Genetic modification of esDC
Fig. 3. Presentation of the endogenously expressed male mH antigen, Dby, by mature esDC. (A) Primary proliferative responses of na R ve, ve, / k femalee T cells from A1xRAG1 femal A1xRAG1 mice, transgenically expressing a TCR specific for the 479–493 epitope of Dby in the context of H2E . T .
cells were cultured mature esDC esDC differentiated from5either karyotypically male ( ) or female ( proliferation by bothwith male and female pulsed with nM Dby479–493 peptide.
E
) ES cells. (B) Stimulation of T cell
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Fig. 4. Comparison of bmDC (A) and esDC (B) genetically modified to express the EGFP reporter gene (filled histograms) under control of the EF1a promoter. Open histograms show levels of autofluorescence among control, nontransfected cells. Lipofection was used in both cases, either to modify terminally differentiated bmDC directly (A) or esDC indirectly by modification and differentiation of a standard ES cell line (B).
Nevertheless, despite the opportunities that adenoviral vectors may provide, their use exacts a heavy price by provoking the premature maturation of DC, the thereby reby sabotaging aspects of their normal physiology [30]. [30].
defici defi cient ent in bo both th al allel leles es of a tar target get gen genee th thro rough ugh homolog hom ologous ous rec recombi ombinati nation. on. Suc Such h an appr approac oach h has already been successfully pursued for the production of lines lines of esDC deficient in expression of Notch 1
Furthermore, the introduction of transgenes and their subsequ sub sequent ent expr express ession ion is acc accompa ompanied nied by the syn syn-thesis of viral proteins whose presentation may elicit vigorous vigo rous CTL res respons ponses, es, ren renderi dering ng the DC the themmselves vulnerable to depletion [29,31,32] depletion [29,31,32].. Whereas esDC share with conventional sources an intrinsi intr insicc res resilie ilience nce to gen genetic etic mod modifi ificati cation, on, we have shown that ES cells may serve as an efficient conduit throug thr ough h wh which ich gen genes es ma may y be int introd roduc uced ed int into o DC without corrupting their function [33] [33].. Since ES cells are comparatively amenable to genetic manipulation and their capacity for self-renewal permits cloning at the si singl nglee-cel celll lev level, el, li lines nes ma may y be pr prod oduce uced d tha that t express a desired mutant phenotype which is faithfully transmitted to esDC, downstream of the differentiation pathway [14,33]. [14,33]. Furthermor Furthermore, e, the capacity for cloning and expansion of individual cells as a means of rescuing rare, mutant phenotypes helps counteract the low tr trans ansduc ductio tion n ef effi ficie ciency ncy of lip lipidid-bas based ed approaches to transfection, thereby avoiding the need for viral vectors and their far-reaching influence on DC fun functio ction. n. Fig. Fig. 4 illustra illustrates tes the eff efficie iciency ncy with which whi ch es esDC DC ma may y be ge gene netic tical ally ly mo modif dified ied thr throug ough h lipof lip ofec ectio tion n of the pa pare rent ntal al ES ce cell ll lin linee (Fi Fig. g. 4B), compare com pared d to ter termin minally ally diff differe erentia ntiated ted bmD bmDC C (Fig. 4A). In addition to paving the way for the rational design of esDC uniformly expressing expressing a desired mutant phenotype, the properties of ES cells may be further explo ex ploite ited d fo forr the pr produ oduct ction ion of li lines nes fu funct nctio ional nally ly
[34], an achievement which is far beyond the scope of [34], tradi tr aditio tional nal str strate ategie giess for gen geneti eticc mo modif difica icatio tion n of terminally termi nally diffe differentiate rentiated d DC.
4. Discu Discussion ssion
ES cells display a number of properties that make them unique, their pluripotency in vitro, capacity for self-renewal and tractability for genetic modification readily distinguishing them from fully differentiated cell types. By exploiting ES cells as a novel source of DC, these features may be harnessed in addition to thos th osee of th thee DC th them emse selv lves es,, to en enha hanc ncee th thei eir r suitability as candidates for immunotherapy. Accordingly ing ly,, esD esDC C may be obta obtained ined in unpa unparall rallele eled d abun abun-dance and with a stability of phenotype that eludes DC differentiated from conventional sources, such as bone marrow progenitors or peripheral blood monocytes. Furthermore, the potential for genetic modific at at io io n o f t he he p ar ar en en ta ta l E S c el el l l in in e p ro ro vi vi de de s oppor op portu tunit nities ies fo forr the ra ratio tional nal des design ign of es esDC DC,, unifor uni formly mly expr expressi essing ng a des desire ired d mut mutant ant phen phenotyp otype, e, aime ai med d at di dire rect ctin ing g th thee ou outc tcom omee of th thee im immu mune ne response upon readministration in vivo. The capacity to obtain such lines without recourse to the use of viral vir al vec vector torss of offe fers rs a sig signif nifica icant nt adv advant antage age ove over r curren cur rentt pra practic ctice, e, sinc sincee the cor corrupt rupting ing inf influen luence ce of viral infection on the physiology of DC is avoided, as
20
P.J. P .J. Fairch Fairchild ild et al. / Internatio International nal Immunop Immunopharmaco harmacology logy 5 (2005) 13–21
are confounding immune responses targeted at viral antigens. The production of a genetically modified ES cell line generates a self-renewing resource that may be cryopreserved long term and differentiated into esDC on de dema mand. nd. Su Such ch a re reso sour urce ce ma may y be dis distr tribu ibute ted d to mult mu ltip iple le ce cent ntre ress so as to en ensu sure re mu much ch-n -nee eede ded d consis con sisten tency cy and rep reprod roduci ucibil bility ity bet betwee ween n cli clinic nical al trials.. Further trials Furthermore, more, by dissoc dissociating, iating, temporally, temporally, the genetic modification of the parent ES cells from the in vivo vi vo adm admini inistr strat ation ion of the re resu sulti lting ng es esDC, DC, am ample ple opportunity is provided for quality control in advance of their clinical use. This may involve identification of th thee co copy py nu numb mber er an and d si site te of in inte tegr grat atio ion n of trans tr ansgen genes es,, as asse sess ssme ment nt of th thee ass assoc ociat iated ed ri risks sks of transf tra nsforma ormation tion and scr screeni eening ng for pote potentia ntiall huma human n pathogens, thereby rigorously addressing fundamental issues of safety. Whereas Wher eas esD esDC, C, gene genetica tically lly mod modifi ified ed to expr express ess v ar ar io io u s c he he mo mo ki ki ne ne s, s, h av av e b ee ee n s uc uc cess cessfully fully employed to enhance vaccination to TAA [35] [35],, their most mo st li like kely ly ap appl plic icat atio ion n li lies es wi with thin in th thee fi fiel eld d of regene reg enerati rative ve med medicin icine, e, for which an acti active ve rol rolee in tolera tol erance nce indu inductio ction n is ess essenti ential. al. Whil Whilee the arr arrest est of esDC at an immature stage of the DC life cycle may favour such a role, genetic modification may also be requir req uired ed to pre prevent vent thei theirr acqu acquisit isition ion of imm immunoge unoge-nicity. As our understanding of the molecular basis of tole tolerog rogenic enicity ity yiel yields ds to inve investig stigatio ation, n, so it may prove feasible to programme into ES cell lines a default propensity for tolerance by their DC progeny. Whil Wh ilee pr prot otoc ocol olss de deve velo lope ped d in th thee mo mous usee aw awai ait t adap ad aptat tatio ion n to hu huma man n ES ce cells lls,, th thee ad adven ventt of es esDC DC of huma human n orig origin in will doub doubtles tlesss pro provide vide sig signif nifican icant t advan ad vance cess ove overr cur curre rent nt app appro roach aches es to DCDC-bas based ed immunot imm unother herapy apy,, her heraldi alding ng an exci exciting ting phas phasee in the realisation of cell replacement therapy within the clinic [36].. [36]
References
[1] Westen estendorp dorp RGJ. Are we becoming less disposable? disposable? EMBO Rep 2004;5:2– 2004;5:2– 6. [2]] Fa [2 Faul ulkn kner er L. Di Dise seas asee ma mana nage geme ment nt:: th thee new new to tool ol fo forr co cost st
[3]
[4] [5]
[6]
[7]
[8]
[9] [10] [11] [1 1]
[12]] [12
[13]
[14]
[15] [16]
Acknowledgements
We are grateful to Drs. Stephen Cobbold and Luis Gracc¸a Gra ¸ a for help helpful ful dis discuss cussions ions and to Pro Prof. f. Rich Richard ard Gardner and Dr. Frances Brook for the provision of ES cell ce ll li line nes. s. Wor ork k in th thee au auth thor ors’ s’ la labo bora rato tory ry wa wass supported by a programme grant from the Medical Research Resear ch Council (UK).
[17] [1 7]
[18]
containment and qualit containment quality y careH careHealth ealth Policy Division. NGA Center for Best Practices; 2003. Thomson JA, Itsko Itskovitzvitz-Eldor Eldor J, Shapir Shapiro o SS, Waknitz Waknitz MA, Swiergiel JJ, Marshalll VS, et al. Embryonic stem cell lines derived derive d from human blast blastocysts ocysts.. Scien Science ce 1998;2 1998;282:1 82:1145– 145– 7. Kellerr G, Snodgrass Kelle Snodgrass HR. Human embryonic embryonic stem cells: the future is now. Nat Med 1999;5:151–2. Lanza RP, RP, Cibelli JB, West MD, Dorff E, Tauer C, Green RM. The ethical reasons for stem cell research. Science 2001; 292: 1299. Soria B, Roche E, Berna G, Leon-Qunito T, Reig JA, Martin F. F. Insuli Ins ulin-s n-secr ecreti eting ng cel cells ls deri derived ved fro from m emb embryon ryonic ic ste stem m cel cells ls normalize glycemia in streptozotocin-induced diabetic mice. Diabetes Diabe tes 2000;49:157 2000;49:157 – 62. Min JY, JY, Yang Y, Converso Converso KL, Liu L, Huang O, Morgan JP, JP, et al. Tr Trans ansplan plantat tation ion of emb embryon ryonic ic ste stem m cel cells ls imp improv roves es cardiacc functi cardia function on in posti postinfarct nfarcted ed rats. J Appl Physiol 2002; 92:288–96. Kim J-H J-H,, Aue Auerbac rbach h JM JM,, Rod Rodrigu riguezez-Go Gomez mez JA, Vela elasco sco I, Gavin D, Lumelsky N, et al. Dopamine neurons derived from embryo emb ryonic nic stem cel cells ls fun functi ction on in an ani animal mal mod model el of Parkinson’s disease. Nature Nature 2002;418:50– 2002;418:50 – 6. Kaufman DS, Thomson JA. JA. Human ES cells—haematopoiesis cells—haematopoiesis and transplantation strategies. J Anat 2002;200:243–8. Steinman RM, Hawi Hawiger ger D, Nusse Nussenzwe nzweig ig MC. Tolerog olerogenic enic dendritic dendri tic cells. Annu Rev Immunol 2003;21:685– 2003;21:685– 711. Fairchild PJ, Wald Fairchild aldman mann n H. Den Dendri dritic tic cel cells ls and pros prospec pects ts for trans transplanta plantation tion tolerance. Curr Opin Immunol 2000;1 2000;12: 2: 528–35. Morell Mor ellii AE, Tho Thomso mson n AW. Den Dendri dritic tic cel cells: ls: reg regula ulator torss of alloimm all oimmuni unity ty and opp opportu ortuniti nities es for tol toleran erance ce ind induct uction ion.. Immunol Rev 2003;196:125–46. Fairchild Fairc hild PJ, Brook FA, Gardne Gardnerr RL, Grac¸a ¸a L, Stron Strong g V, To Tone ne Y, et al. Directed differentiati differentiation on of dendri dendritic tic cells from mouse embryonic embryo nic stem cells. Curr Biol 2000;10:1515 2000;10:1515 – 8. Fairchild Fairc hild PJ, Nolan KF, KF, Waldmann Waldmann H. Probi Probing ng dendritic cell functi fun ction on by gui guidin ding g the dif differ ferent entiat iation ion of emb embryon ryonic ic ste stem m cells. Methods Enzymol 2003;365:169–86. Fong L, Engleman EG. Dendritic Dendritic cells in cance cancerr immunotherimmunotherapy. Annu Rev Immunol 2000;18:245–73. Mayordomo Mayord omo JI, Zorina T, Storkus WJ, Zitvogel L, Celluzi C, Falo LD, et al. Bone marrow marrow-deriv -derived ed dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour antit umour immunity. immunity. Nat Med 1995;1 1995;1:1297– :1297– 302. Zitvoge Zitvo gell L, Ma Mayo yord rdom omo o JI JI,, Tj Tjan andr draw awan an T, De DeLE LEo o AB AB,, Clarke MR, Lotze MT, et al. Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines. J Exp Med 1996;183:87–97. Spechtt JM, Wang G, Do MT Spech MT,, Lam JS, Royal RE, Reeves ME, et al. Den Dendri dritic tic cel cells ls ret retrov rovira irally lly tra transd nsduce uced d wit with h a mod model el
P.J. P .J. Fair Fairchild child et al. / Internati International onal Immunop Immunopharmaco harmacology logy 5 (2005) 13–21
21
antigen gene are therapeutically effective against established pulmonary metastases. J Exp Med 1997;186:1213 – 21. [19] Nestle FO, Alijagic S, Gilliet Gilliet M, Sun Y, Y, Grabbe S, Drummer R, et al. Vaccination of melanoma patients with peptide- or tumor lysate-pulse lysate -pulsed d dendri dendritic tic cells. Nat Med 1998;4:328 1998;4:328 – 32. [20] Thurner B, Haendle I, Roder C, Dieckmann D, Keikavoussi Keikavoussi P, P,
[28]] Irvine [28 Irvine AS AS,, Tr Trind inder er PKE PKE,, Lau Laught ghton on DL, Ket Ketter tering ingham ham H, McDermott RH, Reid SC, et al. Efficient nonviral transfection of dendritic cells and their use for in vivo immunization. Nat Biotechnol Biotec hnol 2000;1 2000;18:127 8:1273 3 – 8. [29]] Mit [29 Mitche chell ll DA, Nai Nairr SK. RNA RNA-tra -transf nsfect ected ed den dendri dritic tic cel cells ls in cancer immunotherapy. J Clin Invest 2000;106:1065–9.
Jonuleit et al.te-derived Vaccination withtic Mage-3A1 peptide-pulsed mature,, N, mature monocyte-de monocy rived dendri dendritic cells expand expands s speci specific fic cytotoxic T cells and induces regression of some metastases in advanced advanc ed stage IV melan melanoma. oma. J Exp Med 1999;190:1669– 1999;190:1669– 78. Nestlee FO, Banchereau J, Hart D. Dendritic cells: Nestl cells: on the move from bench to bedside. Nat Med 2001;7:761–5. Cerundolo Cerundo lo V, V, Hermans IF, Salio M. Dendritic cells: a journe journey y from the laboratory to the clinic. Nat Immunol 2004;5:7–10. Somia N, Verma Verma IM. Gene therapy: trials and tribulations. Nat Nat Rev, Genet 2000;1:91–9. Dhoda Dh odapka pkarr MW MW,, St Stei einma nman n RM RM,, Kra Krasov sovsk sky y J, Mu Munz nz C, Bhardw Bha rdwaj aj N. Ant Antige igen-s n-spec pecifi ificc inh inhibi ibitio tion n of eff effect ector or T cel celll function in humans after injection of immature dendritic cells. J Exp Med 2001;193:233–8. Jonuleit Jonule it H, Schmitt E, Schuler Schuler G, Knop J, Enk AH. Induction Induction + of interl interleukin eukin 10-pro 10-producing ducing,, nonprol nonproliferat iferating ing CD4 T cells with regu regulato latory ry prop properti erties es by rep repetit etitive ive sti stimula mulation tion wit with h
[30] More Morelli lli JM, AE, T Larregin Larr egina aa A Ganste r RW RW, , inant Zahorcha Zaho rchak k irus AF,, AF Plowey Plo wey akayam aka yama T,T,etGan al.ster Recomb Rec ombina nt ade adenov novirus induces maturation of dendritic cells via an NF- nB-dependent pathway. J Virol 2000;74:9617 – 28. [31] Yang Y, Y, Li Q, Ertl HCJ, Wilson JM. Cellular and humora humorall immune responses responses to viral antigens create barriers to lungdirected gene therapy with recombinant adenoviruses. J Virol 1995;69:2004–15. [32] Roth MD, Cheng Q, Harui A, Basak SK, Mitani K, Low TA, TA, et al. Helper-dependent adenoviral vectors efficiently express transgenes in human dendritic cells but still stimulate antiviral immune responses. responses. J Immuno Immunoll 2002;169:4651 2002;169:4651 – 6. [33] Fairc Fairchild hild PJ, Nolan KF, Cartland Cartland S, Grac¸a ¸a L, Waldma aldmann nn H. Stable lines of genetically-modified dendritic cells from mouse embryonic embryo nic stem cells. Tran Transplan splantatio tation n 2003;7 2003;76:606– 6:606– 8. [34] Cheng P, P, Nefedova Y, Y, Miele L, Osbor Osborne ne BA, Gabri Gabrilovic lovich h D. Notch signaling is necessary but not sufficient for differ-
allogeneic immature human dendritic cells. J Exp Med 2000; 192:1213–22. [26] Hawiger D, Inaba K, Drosett Y, Y, Guo M, Mahnke K, Rivera M, et al. Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J Exp Med 2001;194: 769–79. [27]] Ludew [27 Ludewig ig B, Och Ochse senbe nbein in AF AF,, Ode Oderma rmatt tt B, Pau Paulin lin D, Hengartner Henga rtner H, Zinker Zinkernagel nagel RM, et al. Immunotherapy Immunotherapy with dendritic dendri tic cells direct directed ed agains againstt tumor antigens shared with normal host cells results in severe autoimmune disease. J Exp Med 2000;191:795– 2000;191:795– 803.
entiation entia tion of dendri dendritic tic cells. Blood 2003;102:3980– 2003;102:3980– 8. [35] Matsuyosh Matsuyoshii H, Senju S, Hirat Hirataa S, Yoshita oshitake ke Y, Uemura Y, Y, Nishimura Y, et al. Enhanced priming of antigen-specific CTLss in viv CTL vivo o by emb embryo ryonic nic ste stem m cel cell-d l-deri erived ved den dendri dritic tic cells cel ls exp expres ressin sing g che chemok mokine ine alo along ng wit with h ant antige igenic nic pro protei tein: n: application applic ation to antit antitumor umor vacci vaccination nation.. J Immuno Immunoll 2004;1 2004;172: 72: 776–86. [36] Fairchild PJ, Cartland S, Nolan KF, KF, Waldmann H. Embryonic stem ste m cel cells ls and the cha challe llenge nge of tra transp nsplan lantat tation ion tol tolera erance nce.. Trends Immunol 2004;25:465–70.
[21] [22] [23] [24]] [24
[25]
