Type I Interferons Interfere with the Capacity of mrna Lipoplex Vaccines to Elicit Cytolytic T Cell Responses

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1 originl rtile Type I Interferons Interfere with the Cpity of Lipoplex Vines to Eliit Cytolyti T Cell Responses Ans De Beukeler 1, Chrlotte Pollrd 1,2, Sndr Vn Lint 3,4, Kenny Roose 4,5, Lien Vn Hoeke 4,5, Thoms Nessens 1, Viml Kumr Udhykumr 1, Muriel Smet 1, Niek Snders 6, Stefn Lienenklus 7,8, Xvier Selens 4,5, Siegfried Weiss 9,1, Guido Vnhm 3,11, John Grooten 1 nd Stefn De Koker 1,3,4 1 Deprtment of Biomedil Moleulr Biology, Lortory Moleulr Immunology, Ghent University, Ghent, Belgium; 2 Deprtment of Biomedil Sienes, Virology Unit, Institute of Tropil Mediine, Antwerp, Belgium; 3 Deprtment of Biohemistry, Cytokine Reeptor L, Ghent University, Ghent, Belgium; 4 Medil Biotehnology, VIB, Ghent, Belgium; 5 Deprtment of Biomedil Moleulr Biology, Ghent University, Ghent, Belgium; 6 Fulty of Veterinry Mediine, Lortory of Gene Therpy, Ghent University, Ghent, Belgium; 7 Institute for Lortory Animl Siene nd Centrl Animl Fility, Hnnover Medil Shool, Hnnover, Germny; 8 Centre for Experimentl nd Clinil Infetion Reserh, Institute for Experimentl Infetion Reserh, TWINCORE, Hnnover, Germny; 9 Institute of Immunology, Medil Shool Hnnover, Hnnover, Germny; 1 Deprtment of Moleulr Immunology, Helmholtz Centre for infetion Reserh, Brunshweig, Germny; 11 Deprtment of Biomedil Sienes, University of Antwerp, Antwerp, Belgium Given their high potentil to evoke ytolyti T ell responses, tumor ntigen-enoding messenger RNA () vines re now eing intensively explored s therpeuti ner vines. vines lerly enefit from wrpping the into nno-sized rriers suh s lipoplexes tht protet the from degrdtion nd inrese its uptke y dendriti in vivo. Nevertheless, the erly innte host ftors tht regulte the indution of ytolyti T to lipoplex vines hve remined unresolved. Here, we demonstrte tht indue potent type I interferon (IFN) response upon suutneous, intrderml nd intrnodl injetion. Regrdless of the route of immuniztion pplied, these type I IFNs interfered with the genertion of potent ytolyti T ell responses. Most importntly, loking type I IFN signling t the site of immuniztion through the use of n IFNAR loking ntiody gretly enhned the prophylti nd therpeuti ntitumor effiy of in the highly ggressive B16 melnom model. As type I IFN indution ppers to e inherent to the itself rther thn to unique properties of the lipoplex formultion, preventing type I IFN indution nd/or IFNAR signling t the site of immuniztion might onstitute widely pplile strtegy to improve the poteny of vintion. Reeived 24 August 215; epted 28 July 216; dvne online pulition 27 Septemer 216. doi:1.138/mt INTRODUCTION The indution of strong ytolyti CD8 + T ell responses ple of killing trnsformed is onsidered vitl for the suess of therpeuti ner vines. 1 As CD8 + T gurd the intrellulr proteome, their effiient indution typilly requires the presene of ntigens in the ellulr ytosol, where they n enter the lssil route of protesome degrdtion nd mjor histoomptiility omplex (MHC-I) medited ntigen presenttion. In ontrst to protein sed vines, vines sed on messenger RNA () enle protein expression inside the ytosol of trnsfeted nd thus show gret potentil to evoke ytotolyti T ell responses. 2 Due to the limited stility of erly in vitro trnsried (IVT) s, vines hve een predominntly delivered in the formt of ex vivo eletroported dendriti (DCs) for most of the time. 3 Over the pst yers, tehnil improvements in the wy IVT s re prepred (5 Cp modifitions, optimized GC ontent, improved polya tils, stilizing UTRs) hve inresed the stility of IVT s to suh extent protein expression n now e hieved for dys fter diret in vivo dministrtion of the. 4 6 These rekthroughs hve revolutionized the vintion llowing diret injetion of ntigen enoding to e explored for the tretment of ptients with prostte ner, nonsmll lung ell rinom nd melnom When pplied diretly in vivo, vines strongly enefit from wrpping the into nno-sized rriers. Within this ontext, our group previously demonstrted tht ondensing into tioni lipoplexes inreses the poteny of the vine evoked T ell response y severl orders of mgnitude. 14 One of the typil hllmrks of IVT s ondensed into nno-formultions is their pity to eliit intense seretion of Type I interferons (IFNs) in murine nd humn DCs. 14,15 Indeed, IVT ppers to mimi virl RNA in its pity to trigger vriety of ellulr endosoml nd ytosoli RNA sensors tht ll indue signling sde ulminting in the relese of The first two uthors ontriuted eqully to this work. The lst three uthors shre senior uthorship. Correspondene: Stefn De Koker, Deprtment of Biomedil Moleulr Biology, Lortory Moleulr Immunology, UGent- VIB Reserh Building, FSVM L of Moleulr Immunology Tehnologieprk, Gent, Belgium. E-mil: stefn.dekoker@vi-ugent.e vol. 24 no. 11, nov. 216

2 Totl flux p/s Enhning Cner Vintion Effiy type I IFNs Type I IFNs re highly pleiotropi ytokines tht n either promote or inhiit T ell responses dependent on the ontext. Type I IFNs n ugment T ell immunity y tivting DCs nd inresing ntigen presenttion. Conversely, the ntivirl tions of type I IFN, prodution of RNAses nd instigtion of trnsltion rrest might interfere with the expression of the enoded ntigen nd therefore negtively impt T ell immunity. Type I IFN signling on ntigen experiened T n promote T ell prolifertion, survivl, nd differentition into effetor. 18 Nevertheless, type I IFN exposure prior to T ell reeptor tivtion n indue ntiprolifertive nd poptoti progrmmes in T How type I IFNs impt the hrteristis of the T ell responses to lipoplex vines nd their effiy to ontrol tumor growth is therefore fr from forgone onlusion nd onstitutes the min gol of this study. Using n IFN-β reporter mouse strin, we were le to demonstrte tht instigte profound type I IFN responses upon suutneous, intrderml, nd intrnodl injetion. In shrp ontrst to the enefiil role of type I IFNs in protein nd peptide sed vines, type I IFNs severely hmpered priming of vine speifi T ell responses nd the genertion of ntitumor immunity to lipoplex sed vintion. Preventing type I IFN indued signling through odministrtion of n IFNAR loking ntiody t the site of sed vintion mplified the ytotolyti T ell response nd signifintly strengthened vine eliited tumor ontrol in prophylti nd therpeuti settings. RESULTS indue potent type I IFN response in vivo Ctioni liposomes hve een reported to inrese T ell responses to enoded ntigens. 26 In this study, liposomes omposed of the tioni lipid 1,2-dioleoyl-3-trimethylmmonium-propne (DOTAP) nd the helper lipid 1,2-dioleoylsn-glyero-3-phosphoethnolmine (DOPE) were used to ondense into lipoplexes. Preliminry reserh ws done to determine the nitrogen/phosphte rtio most suited for in vivo pplition nd is shown s dditionl dt (see Supplementry Figure S1). We evluted two N/P rtios tht give yield to lipoplexes of similr size (± 3 4 nm) ut opposite hrge, nmely lipoplexes t N/P1 hd negtive zet-potentil of 18 mv nd N/P1 lipoplexes displyed positive hrge of +32 mv (see Supplementry Figure S1,). Further, we ddressed lipoplexes of rtio N/P1 s most suited to yield high expression levels of the delivered (see Supplementry Figure S1) nd to indue proper indution of IFN-ɣ produing CD8 + nd CD4 + T upon suutneous injetion (see Supplementry Figure S1d). As onsequene, N/P1 ws seleted in ll further experiments imed t ddressing the impt of type I IFNs on the effiy of to yield T ell immunity. Previously, we hve demonstrted tht DOTAP-sed lipoplexes eliit strong type I IFN seretion upon inution with one mrrow derived DCs in vitro. 14 To ddress to whih extent would trigger type I IFNs in vivo upon suutneous injetion, we used n IFN-β reporter mouse in whih firefly luiferse enoding sequene hs een pled under the ontrol of the IFN-β promoter (Figure 1). 27 As type I IFN prodution is regulted y self-enforing feedforwrd loops, heterozygous reporter mie (IFN-β +/Δβ-lu ) were used to llow signl mplifition y erly indued IFN-β. Mie were injeted suutneously with respetively DOTAP liposomes (no ), unformulted or. In vivo ioluminesene imging reveled strong indution of the IFN-β promoter to injetion of nked nd of, ut not to liposomes without (Figure 1,). Strikingly, nked ovlumin (OVA) eliited the most prominent indution of type I IFNs, lerly inditing tht type I IFN indution to is inherent to the itself rther thn to unique fetures of the. Type I IFNs impt the mgnitude nd funtionl hrteristis of the vine eliited CD8 + T ell response Depending on the ontext, type I IFNs hve een reported to either promote or interfere with the genertion of T ell responses. As onsequene, we thoroughly ddressed the impt of type I IFN signling on the mgnitude nd funtionlity of the T ell response generted y lipoplex vintion through omprtive immuniztion studies in wild type mie nd in mie IFN-β promoter liposomes LoxP My-tg n.s. Luiferse liposomes SV4 PolyA Figure 1 indue potent type I IFN response in vivo. () Grphil sheme of the IFN-β reporter onstrut. The my-tgged luiferse gene is rought under the ontrol of the IFN-β promoter y the Cre-Lox system. (,) IFN-β +/Δβ-lu mie were suutneous (s..) injeted with 1 µg of OVA, nd liposomes. Luminesene ws mesured 6 hours postinjetion. Dt re shown s men ± SD of four mie. P <.1. P <.5 (Mnn Whitney test). = 5% gluose wter; liposomes = DOTAP/DOPE lipids; lipoplexes = messenger RNA omplexed to liposomes., messenger RNA; IFN, interferon, OVA, ovlumin; SD, stndrd devition. Moleulr Therpy vol. 24 no. 11 nov

3 Enhning Cner Vintion Effiy lking he ommon IFN-α/β reeptor IFNAR1 ( ). First, we ddressed the effets of type I IFNs on the initil priming of ntigen-speifi T. To this end, roxyfluoresein diette suinimedyl ester (CFSE) leled trnsgeni OVA-speifi CD8 + T (OT-I T ) were trnsferred to respetively wild type nd mie, whih were susequently immunized with OVA. Four dys postimmuniztion, the drining poplitel lymph nodes were disseted nd OT-I T ell prolifertion ws nlyzed y flow ytometry (Figure 2). As shown in Figure 2, mie showed strongly elevted OT-I prolifertion when ompred with wild type mie. This negtive impt of type I IFNs on the mgnitude of the vine evoked CD8 + T ell response ws onfirmed y quntifition of vine eliited OVA-speifi CD8 + T in the lood of wild type versus mie (Figure 2). Five dys fter immuniztion, OVAspeifi CD8 + T were hrdly detetle in the lood of wild type mie ut rehed up to 3% of ll CD8 + T in the lood of mie. No signifint numers of OVA-speifi T were deteted in response to unformulted OVA. Next, we nlyzed the impt of IFNAR defiieny on the funtionl properties of the vine indued CD8 + T ell response. As type I IFNs hve een reported to stimulte the differentition of primed CD8 + T into effetor, 2,21,28,29 the inresed numers of vine eliited CD8 + T ell response oserved in mie not neessrily trnslte into inresed effetor funtion in these mie. To ddress this issue, we ompred OVA-speifi IFN-γ seretion nd trget ell speifi lysis etween immunized wild type nd mie. Enzyme-linked immunosorent spot (ELISPOT) ssys were performed on splenoytes 2 weeks fter the ooster immuniztion with OVA to quntify the numers of IFN-γ produing OVA-speifi T. As depited in Figure 2d, immunized mie showed strong inrese in the numers of OVA-speifi IFN-γ sereting T. The ytolyti pity of the evoked CD8 + T ell response ws nlyzed through n in vivo killing ssy. In rief, 2 weeks fter ooster immuniztion with OVA, mie were hllenged with 1:1 rtio of OVA peptide-pulsed CFSE hi splenoytes (trget ) nd nonpulsed CFSE low splenoytes (nontrget ). After 2 dys, spleens were disseted nd the rtio of trget versus nontrget ws nlyzed y flow ytometry to determine the extent of killing of the trget. Wheres immuniztion of wild type mie with OVA resulted only in limited killing of the trget, virtully ll trget were eliminted in immunized mie (Figure 2e f). Tken together, these dt lerly demonstrte tht IFNAR defiieny inreses initil T ell priming to suutneously dministrted lipoplex vines nd tht type I IFN re not required for these ntigen-experiened T to quire effetor funtion. As we oserved previously, n inrese in the expression of lipoplex delivered in one mrrow derived DCs lking IFNAR, we deided to quntify expression fter suutneous injetion of in wild type nd mie. If mie would show strongly inresed expression levels, inresed ntigen expression might well underlie the rise in initil T-ell prolifertion we oserved in mie. To ddress this issue, luiferse enoding ws ondensed into lipoplexes t N/P1 nd luiferse expression ws ssessed through in vivo ioluminesene mesurement. Although luiferse expression ws slightly elevted in the IFNAR defiient setting, this inrese ws very sutle nd did not rehed signifine (see Supplementry Figure S2). As onsequene, events downstrem of ntigen expression must e t the origin of the drmtilly rised T ell responses in mie. Impt of type I IFNs on the effiy of ntitumor immunity eliited y lipoplex vintion The funtionl impt of type I IFNs on ntitumor immunity medited y lipoplex vintion ws ddressed in the highly ggressive B16.OVA melnom model. Mie were either vinted prophyltilly or therpeutilly ording to the shedule shown in Figure 3,d. In wild type mie, prophylti vintion signifintly inresed the medin survivl time from 17 to 29 dys (Figure 3). In line with their elevted vine eliited T ell responses, mie enefited even more from vintion thn wild type mie, s the medin survivl time inresed from 14 to 4 dys (Figure 3). This oservtion is highly striking s mie notoriously lk spontneous ntitumor immune responses nd suum muh fster to tumors when left untreted Therpeuti vintion used smll though nonsignifint improvement in medin survivl time from 34 to 47 dys in wild type mie (Figure 3e). Conversely, therpeuti vintion yielded signifint survivl enefit in mie with n inrese in medin survivl time from 2 to 35 dys (Figure 3f). Nevertheless, in the therpeuti vintion setting, vinted wild type mie still ontrolled tumors etter thn vinted mie, feture tht n e most likely sried to the lk of spontneous ntitumor responses in the IFNAR defiient setting. Antiody medited IFNAR lokde improves the effiy of the vine evoked ntitumor immune response Results of the experiments in the previous prgrph illustrte tht in immunized wild type mie tumor growth ontrol is determined y the omined strength of the spontneous nd vine eliited immune responses, wheres in mie tumor ontrol will entirely depend on the vine eliited immune response. As onsequene, diret omprisons of tumor growth rtes etween immunized wild type nd mie do not llow relile ssessment of the impt of type I IFNs on vine medited tumor ontrol. To irumvent the detrimentl effet of geneti IFNAR defiieny on spontneous ntitumor immunity, we therefore, deided to swith to ntiody medited inhiition of IFNAR signling t the spot of vintion in wild type mie. Lol interferene with IFNAR signling should leve the spontneous ntitumor response intt, nd therey llow us to speifilly ddress the impt of type I IFN signling on vine medited tumor ontrol. First, we vlidted whether ntiody-medited IFNAR lokde would indeed mplify the CD8 + T ell response eliited y the lipoplex vine in wild type mie. As n e ppreited from Figure 4, oinjetion of the IFNAR loking ntiody inresed the prolifertion of OVA-speifi OT-I in response to, while the isotype mthed ntiody hd no vol. 24 no. 11 nov. 216

4 IFN-γ SFC/1 6 SSC OVA-PE CD3 Enhning Cner Vintion Effiy FSC Cells SSC-H Single SSC-A % OT-I divided CD3+CD19 CD19 Tet+CD8+ Live/ded Count Alive FSC % OT-I divided % OVA -speifi CD8 + T d 8 6 CD8 FITC e f % speifi killing Trget Trget Trget Trget CFSE Figure 2 Type I IFNs impt the mgnitude nd funtionl hrteristis of the vine eliited CD8 + T ell response. () Gting strtegy used for OVA- speifi CD8 + T ell ounting nd prolifertion. Cells re gted sed on FSC nd SCC, efore single re gted sed on SSC-re nd height. Living re seleted nd gted for CD3 + CD19 - T. Within CD8 + T, OVA-speifiity is gted y leling with MHC-I SIINFEKL PE dextrmer. Prolifertion of CFSE positive OVA-speifi CD8 + T is shown. () Two dys prior to immuniztion CFSE-leled OT-I were doptively trnsferred to wild type () nd mie. Suutneous (s..) immuniztion ws performed t til se with 1 µg OVA, nked or liposomes lone. Four dys fter immuniztion inguinl lymph nodes were isolted nd CD8 + T ell prolifertion ws nlyzed y flow ytometry. Dt re shown s men of 2 3 mie. P <.1 (Chi-squre test). () Wild type () nd mie were s.. injeted with 2 µg OVA or nked OVA s ontrol. Blood ws isolted 5 dys lter nd the perentge OVA-speifi CD8 + T ws determined y dextrmer stining followed y flow ytometry. Dt re shown s men of four mie per group. P <.1 (Chi-squre test). (d) Wild type () nd mie were immunized s.. with 2 µg OVA or nked s ontrol. After 2 weeks, mie were oosted with the sme formultion. Spleens were isolted 2 weeks fter the oost immuniztion, nd the numer of OVA-speifi interferon-γ spot-forming CD8 + nd CD4 + T (SFC) ws determined y enzyme-linked immunosorent spot (ELISPOT). Dt re shown s men of 2 4 mie per group. P <.1 (Chi-squre test). (e,f) Wild type () nd mie were immunized with two-week intervl with nked OVA or OVA lipolexes. Two weeks fter the oost immuniztion, mixture of CFSE-leled pulsed with ontrol (CFSE low ) or OVA peptide (CFSE high ) were doptively trnsferred. Speifi killing ws mesured 2 dys lter y flow ytometry. Dt re presented s mens of 1 1x ((CFSE high / CFSE low ) immunized mie / (CFSE high / CFSE low ) mok-mie ) of 3 4 mie per group. P <.1 (Chi-squre test). = OVA-oding messenger RNA; = messenger RNA omplexed to DOTAP/DOPE liposomes. CFSE, roxyfluoresein diette suinimedyl ester;, messenger RNA; IFN, interferon, OVA, ovlumin; SD, stndrd devition. Moleulr Therpy vol. 24 no. 11 nov

5 Enhning Cner Vintion Effiy Tumor growth Week Prime Week 2 Week 4 1 B16.OVA Dys post-tumor hllenge Dys post-tumor hllenge d Tumor growth Dy 75 B16.OVA Dy 4 Dy 6 e ns f Dys post-tumor hllenge Dys post-tumor hllenge Figure 3 Impt of type I IFNs on the effiy of ntitumor immunity eliited y lipoplex vintion. () Prophylti vintion sheme. Wild type () mie () nd mie () were either mok s.. immunized (i.e., injeted with only) or immunized with 2 μg of. After 2 weeks, mie were oosted with the sme formultion. At week 4, mie were inoulted with 1, OVA-expressing B16 melnom. (n = mie/group). (d) Therpeuti vintion sheme. Wild type () mie (e) nd mie (f) were inoulted with 75, B16.OVA melnom. After 4 nd 6 dys, immuniztion ws performed with similr preprtions s in the prophylti setting. (n = 5 6 mie/group). = OVA-oding messenger omplexed to DOTAP/DOPE liposomes. P <.1; P <.1; P <.1 (Mntel-Cox log-rnk test)., phosphte-uffered sline;, messenger RNA; IFN, interferon, OVA, ovlumin; SD, stndrd devition. impt on OT-I prolifertion. Next, we determined if loking IFNAR t the site of immuniztion would improve the ntitumor effiy of the lipoplex vines in se of prophylti (Figure 4,d) nd therpeuti vintion (Figure 4e,f). In the prophylti vintion setting, oinjetion of the IFNARloking ntiody MAR1-5A3 with the OVA signifintly improved the survivl rte of immunized mie (Figure 4d). Importntly, the enefit of loking IFNAR ws preserved in the therpeuti vintion setting, s mie immunized with in the presene of MAR1-5A3 displying n improved outome ompred with mie reeiving the sme vine lone or omined with n isotype ontrol ntiody (Figure 4f). Tken together, these findings demonstrte tht type IFNs indued y lipoplex vines negtively impt the vine eliit T ell response nd its effiy to ontrol tumor growth upon suutneous vintion. Type I IFNs dmpen ytolyti T ell responses to intrderml nd intrnodl lipoplex vintion As the route of immuniztion hs drmti impt on the type of innte immune the enounter nd therey potentilly lso on the ensuing T ell response, we deided to evlute the impt of type I IFNs on the ytolyti T ell response vol. 24 no. 11 nov. 216

6 Enhning Cner Vintion Effiy OT-I divided 1 Count % OT-I divided FITC + iso ontr. + IFNAR A + iso. ontr. + IFNAR A e Tumor growth Tumor growth Week Prime Week 2 Week 4 1 B16.OVA Dy 75 B16.OVA Dy 4 Prime Dy 9 d 1 f Dys fter B16.OVA inoultion Dys fter B16.OVA inoultion + iso. ontr. + IFNAR A + iso. ontr. + IFNAR A Figure 4 Antiody-medited loking of IFNAR improves the effiy of the vine evoked ntitumor immune response. (,) Two dys prior to immuniztion CFSE-leled OT-I were doptively trnsferred to wild type () mie. Immuniztion ws performed in the footpd with 1 µg in the sene or presene of 2 µg IFNAR loking ntiody or isotype ontrol. Four dys fter immuniztion inguinl lymph nodes were isolted nd CD8 + T ell prolifertion ws nlyzed y flow ytometry. Dt re shown s men of 3 6 mie per group. P <.1 (Chi-squre test). () A representtive smple out of 3 6 mie eh group is presented. () Prophylti vintion sheme. (d) Wild type () mie were immunized s. with 2 μg of in sene or presene of 2 µg of the IFNAR loking ntiody or isotype ontrol. After 2 weeks, mie were oosted with the sme formultion. At week 4, mie were inoulted with 1, B16.OVA melnom (n = 6 8 mie/group). P <.5 (Mntel-Cox log-rnk test). (e) Therpeuti vintion sheme. (f) Wild type () mie were inoulted with 75, B16.OVA melnom. After 4 nd 9 dys, immuniztion ws performed using 2 μg of in sene or presene of the IFNAR loking ntiody or isotype ontrol (2 µg) (n = 6 8 mie/group). P <.5 (Mntel-Cox log-rnk test). = OVA- oding messenger omplexed to DOTAP/DOPE liposomes. CFSE, roxyfluoresein diette suinimedyl ester;, messenger RNA; IFN, interferon, OVA, ovlumin; SD, stndrd devition. to intrderml nd intrnodl immuniztion with. lso instigted profound type I IFN response to intrderml (Figure 5) nd intrnodl (Figure 5) injetion. In terms of T ell immunity, intrderml immuniztion with ehved muh like suutneous immuniztion, with the strength of the ytolyti T ell response shifting from ner sent in wild type mie to virtully omplete in mie (Figure 5). In line with reports of the Thielemns 32 nd Shin 33 groups, intrnodl immuniztion turned out to e y fr the most potent route of immuniztion with strong ytolyti T ell responses now eing evident in immunized wild type mie (Figure 5d). Nevertheless, even intrnodl immuniztion ws ided y IFNAR defiieny, s the ytolyti T ell response ws even further enlrged in mie. Tken together, these dt firmly demonstrte tht type I IFNs dmpen the strength of the ytolyti T ell response evoked y lipoplex-sed vintion, regrdless of whether the re delivered suutneous, intrderml or intrnodl. Moleulr Therpy vol. 24 no. 11 nov

7 Totl flux (p/s) Totl flux (p/s) Enhning Cner Vintion Effiy 1 6 lipoplexes lipoplexes Figure 5 Type I IFNs inhiit the indution of ytolyti T regrdless of the route of immuniztion. () IFN-β +/Δβ-lu mie were intrdermlly injeted with 1 µg of OVA omplexed or. In vivo ioluminesene ws mesured 6 hours postinjetion. Dt re shown s men ± SD of three mie. P <,1 (t-test). () Wild type () nd mie were immunized with two-week intervl with 1 µg of. Two weeks fter oost immuniztion, mixture of CFSE-leled pulsed with ontrol (CFSE low ) or OVA peptide (CFSE high ) were doptively trnsferred. Speifi killing ws mesured fter 2 dys y flow ytometry. Killing perentges were lulted with the following formul: 1 1x ((CFSE high /CFSE low ) immunized mie /(CFSE high / CFSE low ) mok-mie ) of five mie per group. P <.1 (t-test). () IFNβ +/Δβ-lu mie were intrnodlly injeted with 1 µg of OVA or mok treted. In vivo ioluminesene ws mesured 6 hours postinjetion. Dt re shown s men ± SD of three mie. P <.1 (t-test). (d) Wild type () nd IFNAR / mie were immunized with two-week intervl with 1 µg of OVA nd killing ws performed s previously desried. P <.5 (t-test). = OVA- oding messenger omplexed to DOTAP/DOPE liposomes. CFSE, roxyfluoresein diette suinimedyl ester;, messenger RNA; IFN, interferon, OVA, ovlumin;, phosphte-uffered sline; SD, stndrd devition. DISCUSSION Condensing into lipoplexes signifintly improves the strength of the T ell response ginst the enoded ntigen upon in vivo immuniztion. Nevertheless, the key innte host ftors tht determine the poteny of lipoplex vines nd their effiy to instigte ntitumor immunity hve remined unresolved. Erlier, we hve shown tht type I IFNs re the most prominent ytokines sereted y DCs when inuted with. 14,15 As type I IFNs re mjor regultors of T ell immunity to viruses nd to tumors, we deided to ddress their funtionl impt on the T ell response to lipoplex d % Speifi killing % Speifi killing vines. Vintion studies in mie reveled drmtilly inresed priming of vine speifi T in the sene of IFNAR signling. These vine primed T quired full effetor funtion nd effiiently eliminted trget. When hllenged with the highly ggressive B16 melnom model, vinted mie enefited more from lipoplex vintion ompred with wild type mie in terms of inrese in survivl time to nontreted ontrols. Nevertheless, therpeutilly vinted mie still suumed erlier to B16 hllenge when ompred with vinted wild type mie. mie however lk spontneous ntitumor immunity, mking diret omprisons etween en wild type mie onerning the effets of vintion on tumor ontrol diffiult to interpret. To void ny onfounding effets of geneti IFNAR defiieny on spontneous versus vine eliited ntitumor immunity, we therefore shifted to odministrtion of n IFNAR loking ntiody t the time nd spot of immuniztion in wild type mie. Bloking IFNAR t the vintion site onferred sustntil survivl enefit in response to oth prophylti nd therpeuti vintion, therey estlishing type I IFNs s host ftors tht severely hmper the effiy of s ntitumor vines. The ext mehnism y whih type I IFNs exert their negtive impt remins lrgely unresolved. Type I IFNs n ffet the instigtion of effetor T ell immunity t multiple levels. First, s type I IFNs re potent ntivirl ytokines tht typilly tivte RNAses nd lok trnsltion to prevent virl replition, 34 they might hmper T ell immunity to vines y lowering the mount of ntigen expressed, feture we hve reported on using in vitro BM-DCs inuted with. 14 Nevertheless, the impt of IFNAR defiieny on the expression level in vivo ws very limited nd thus most likely does not onstitute the mjor ftor ehind the drmtilly improved ytolyti T ell response in mie. A potentil explntion is tht type I IFNs exert their negtive impt diretly t the level of the T ell. Indeed, wheres type IFNs n lerly t s signl 3 ytokines tht promote the differentition of ntigen primed CD8 + T into ytolyti effetors, 29 they n lso lok T ell prolifertion nd even instigte T ell poptosis Whih of these opposing effets previls, depends on the kinetis of T ell exposure to type I IFNs. 17 If IFNAR triggering preedes T-ell reeptor triggering, the T ell inhiitory properties previl. In se of lipoplex vintion, type I IFN relese ours rpidly, TLRs nd other RNA sensing reeptors n e triggered in the endosoml omprtments even efore the leves the endosomes for trnsltion, nd most likely efore DCs tht hve tken up the hve rehed the lymph nodes to present the ntigen. Nevertheless, studies using mie seletively defiient in IFNAR in DCs or in T re required to shed further light t whih stge type I IFNs extly interfere with T ell immunity to. In generl, our findings regrding the negtive impt of type I IFNs on T ell immunity to lipoplex vines re in sheer ontrst with two reent reports y Krnz 35 nd Broos. 36 Although speultive, we elieve tht these disrepnies n e lrgely ttriuted to the different route (intrvenous) of vine delivery pplied in these studies. Intrvenous injetion of vol. 24 no. 11 nov. 216

8 Enhning Cner Vintion Effiy will result in different ell types trgeted nd in n ltered kinetis of ntigen expression nd type I IFN indution. As explined ove, the stimultory versus inhiitory effets of type I IFNs on T ell immunity lrgely depend on the timing of T ell exposure to type I IFNs. Intrvenous injetion of therey might result in n improved onvergene of ntigen expression nd type I IFN indution, using the enefiil effets of type I IFNs to previl. In summry, we hve firmly estlished type I IFNs s host ftors tht negtively regulte the pity of lipoplex vines to instigte ytolyti T upon suutneous, intrderml, nd intrnodl dministrtion. As type I IFN indution is inherent to IVT s, our findings re of importne to mny other nno-formultions explored for vintion. If so, strtegies to prevent or redue type I IFNs might e of gret vlue to improve the linil effiy of vines. Additionl experimentl hrteriztion dt re provided in Supplementry Dt. MATERIALS AND METHODS Mie. Femle wild type C57BL/6 mie were purhsed from Jnvier (Le Genest Sint Isle, Frne). OT-I mie rrying trnsgeni CD8 + T ell reeptor speifi for the MHC I-restrited OVA peptide SIINFEKL were donted y Brt Lmreht from Ghent University (Ghent, Belgium). Ifnr1 / mie were red t the reeding fility of the Vlms Instituut voor Biotehnolgoy (VIB, Ghent, Belgium). C57BL/6 luiferse reporter mie (IFN-β +/Δβ-lu ) were red t the Helmholtz Center for Infetion Reserh (HZI). All mie were 7 12 weeks old t the strt of the experiment nd mintined under speifi pthogen-free onditions. Animls were treted ording to the Europen guidelines for niml experimenttion. All experiments were pproved y the lol ethil ommittee for niml experiments of Ghent University (Ghent, Belgium) or of the Helmholtz Center for Infetion Reserh (Brunshweig, Germny). Prodution of in vitro trnsried. The pgem4z-ova-a64 nd the pgem4z-egfp-a64 plsmids were kindly donted y Dvid Bozkowski from Duke University (Durhm, NC). The pbluesript-lu- A64 plsmid ws provided y Jonn Rejmn from Ghent University (Ghent, Belgium). All plsmids were propgted in E. oli ompetent (Strtgene, L Joll, CA) nd purified using endotoxin-free QIAGENtip 5 olumns (Qigen, Chtsworth, CA). The pgem4-ova-a64 nd pgem4z-egfp-a64 plsmids were linerized with SpeI (MBI Ferments, St Leon-Rot, Germny), wheres the pbluesirpt-lu-a64 plsmid ws linerized with DrI (MBI Ferments). Linerized plsmids were purified using PCR purifition kit (Qigen, Venlo, The Netherlnds) nd RNA ws trnsried using the T7 mmessge Mhine Kit (Amion, Austin, TX) ording to the mnufturer s instrutions. The in vitro trnsried ws purified y lithium hloride preipittion. Immuniztions nd injetions of. Suutneous immuniztions were performed in C57BL/6 mie twie t til se in twoweek intervl. Aording to the experiment 1 or 2 μg of OVA-enoding ws omplexed with DOTAP/DOPE lipids in N/P rtio of 1 (Avnti Polr Lipids, Alster, AL) nd injeted in totl volume of 4 μl of 5% gluose wter (Amion, Thermo Fisher, Wlthm, MA). For intrnodl delivery of, C57BL/6 mie were nesthetized with ketmine (7 mg/kg; Cev) nd xylzine (1 mg/kg; Byer). The inguinl lymph node ws surgilly exposed nd injeted with 1 µg RNA lipoplexes in totl volume of 15 µl. Susequently, the wound ws losed. For intrderml immuniztion, 1 µg of ws injeted into the er dermis in totl volume of 2 µl. Aordingly to the experiments, the totl vine volume inluded 2 μg of MAR1-5A3 (ntimouse IFNAR) or mouse IgG1 isotype ontrol (oth from Leino Tehnologies, St. Louis, MO). For in vivo mesuring of expression levels wild type nd mie were injeted s.. with 1 µg of luiferse enoded. expression levels were mesured 8 hours fter injetion vi in vivo iolumenesene. Flow ytometry. All flow ytometri experiments were performed on triple-lser (B-V-R) LSR-II (Beton Dikinson, Sn Jose, CA) nd nlyzed with FlowJo (Treestr, Ashlnd, OR). Cells were stined with α-cd16/ CD32 (BD Biosienes, Sn Diego, CA) to lok nonspeifi FR inding, nd with Live/Ded Fixle Aqu stin (Invitrogen, Mereleke, Belgium) to eliminte ded from nlysis. Antiodies used re α-cd8 PerCP, α- CD3 pifi lue, α-cd19 APC-Cy7, α-cd11 PerCP-Cy5.5, α-f4/8 APC (ll BD Biosienes), nd MHC dextrmer H-2 K/SIINFEKL-PE (Immudex, Copenhgen, Denmrk). In vivo imging of IFNβ indution. Heterozygous luiferse reporter mie (IFN-β +/Δβ-lu ) were injeted suutneously, with phosphte-uffered sline (), 1 µg of OVA- omplexed with DOTAP/DOPE liposomes t n N/P rtio of 1 (Avnti Polr Lipids), DOTAP/DOPE lone or nked OVA- in totl volume of 2 μl 5% gluose wter. Intrderml or intrnodl injetions were performed with 1 µg of t n N/P rtio of 1 (Avnti Polr Lipids), DOTAP/DOPE lone or nked OVA in totl volume of 1 2 μl 5% gluose wter. IFNβ indution ws mesured t, 3, nd 6 hours fter injetion vi in vivo iolumenesene. In vivo ioluminesene imging. For in vivo imging, mie were injeted intrvenously with 15 mg/kg of D-luiferin (PerkinElmer, Wkthm, MA) in nd monitored using n IVIS lumin II imging system. Photon flux ws quntified using the Living Imge 4.4 softwre (ll from Cliper Life Sienes, Hopkinton, MA). Enzyme-linked immunosorent spotc57bl/6 mie were immunized twie with 2 µg of DOTAP/DOPE-omplexed OVA-enoding in two-week intervl. Two weeks fter the oost immuniztion, spleens were isolted nd pssed through 7 μm nylon striners (BD Biosienes) to otin single ell suspensions. Red lood were lysed using ACK red lood ell lysis uffer (BioWhittker, Wkersville, MD) nd were ultured for 24 hours on IFN-γ (Dilone, Besnçon, Frne) preoted 96-well pltes in the presene of 1 μg/ml OVA peptides (Anspe, Fremont, CA). To quntify the mount of OVA-speifi CD8 + nd CD4 + T we pulsed the splenoytes with respet to 1 µg/ml MHC-I nd MHC-II OVA peptides. Spots were nlyzed ording to the mnufturer s instrutions using enzyme-linked immunosorent spot (ELISPOT) reder. CD8+ T ell dextrmer stining. Mie were immunized twie with 2 µg of DOTAP/DOPE omplexed OVA-enoding s desried previously. After 5 dys, lood smples were tken nd red lood were removed using ACK lysis uffer (BioWhittker). Cells were stined with α-cd16/cd32 (BD Biosienes), Live/Ded Fixle Aqu stin (Invitrogen), α-cd8 PerCP, α- CD3 pifi lue, α-cd19 APC-Cy7 (ll BD Biosienes) nd MHC dextrmer H-2 K/SIINFEKL-PE (Immudex). In vivo T ell prolifertion ssy. Two dys efore immuniztion OT-I were leled with 5 µmol/l CFSE (Invitrogen). Two million CFSE-leled OT-I were i.v. injeted into wild type nd mie 2 dys efore immuniztion. Immuniztion ws performed s previously desried. Four dys fter immuniztion drining lymph nodes were isolted nd CD8 + T ell division ws nlyzed y flow ytometry. Cells were stined with α-cd16/cd32 (BD Biosienes), Live/Ded Fixle Aqu stin (Invitrogen), α-cd8 PerCP, α- CD3 pifi lue, α-cd19 APC-Cy7 (ll BD Biosienes), nd MHC dextrmer H-2 K/SIINFEKL-PE (Immudex). In vivo killing ssy. Splenoytes from femle wild type mie were pulsed with 1 µg/ml of MHC-I OVA peptide or HIV-1 Gg peptide s ontrol efore leling with 5 µmol/l or.5 µmol/l CFSE (Invitrogen), respetively. Moleulr Therpy vol. 24 no. 11 nov

9 Enhning Cner Vintion Effiy Leled were mixed t 1:1 rtio, nd totl of mixed were doptively trnsferred into immunized mie 2 weeks fter oost. Splenoytes from host mie were nlyzed 2 dys lter y flow ytometry fter stining with α-f4/8 (BD Biosienes) to exlude uto-fluoresent mrophges. Perentge ntigen-speifi killing ws determined using the following formul: 1 1 ((% CFSE hi / % CFSE low immunized mie ) /(% CFSE hi / % CFSE low ) nonimmunized mie. Tumor hllenge. For the prophylti tumor experiments, immunized mie were inoulted s.. in the flnk with 1 5 B16-OVA melnom (VIB ell nk) in 2 µl 2 weeks fter oost immuniztion. Immuniztions were performed s desried ove. Tumor growth ws followed y mesuring the tumor size index, i.e., the produt of the lrgest perpendiulr dimeters, with liper. For ssessment of therpeuti effiy, B16-OVA melnom in 2 µl were dministered 4 dys prior to immuniztion. immuniztions were given 2 5 dys fter priming. SUPPLEMENTARY MATERIAL Figure S1. Chrteriztion of t nitrogen/phosphte (N/P) rtio 1 nd 1. Figure S2. Luiferse expression ws mesured in wild type () en mie fter s. injetion of 1 µg luiferse enoding t rtio N/P1. Supplementry Dt. ACKNOWLEDGMENTS This work ws supported y grnts from the Interuniversitry Attrtion Pole onsortium IUAPVII/63-12C7812W-B/12917/1IUAP, the UGhent Conerted Reserh Consortium BOF12/GOA-B/12424/1, the Fund for Sientifi Reserh Flnders projet G.226.1, the Strtegi Bsi Reserh Progrm of the Flnders government projet 816, the Europrise (Europen vines nd miroiides Enterprise) projet 37611, the UGhent Methuslem projet BOF9/1M79, nd the SOFI-B (Seondry Reserh Funding) t the Institute of Tropil Mediine. De B.A. knowledges I (Agentshp voor innovtie door wetenshp en tehnologie, Vlnderen) for PhD sholrship. Pollrd C. ws supported y PhD sholrship from SOFI-B. Roose K. is supported y EC FP7 projet FLUNIVAC. De K.S. knowledge FWO Flnders for funding nd Vn Lint S. knowledge BOF- UGhent. 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