Dendritic cells engineered to secrete anti-dcr3 antibody augment cytotoxic T lymphocyte response against pancreatic cancer in vitro

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1 Submit Mnuscript: Help Desk: DOI: /wjg.v23.i5.817 World J Gstroenterol 2017 Februry 7; 23(5): ISSN (print) ISSN (online) 2017 Bishideng Publishing Group Inc. All rights reserved. ORIGINAL ARTICLE Bsic Study Dendritic cells engineered to secrete nti-dcr3 ntibody ugment cytotoxic T lymphocyte response ginst pncretic cncer in vitro Jing Chen, Xio-Zhong Guo, Hong-Yu Li, Ji-Jun Zho, Wen-D Xu Jing Chen, Xio-Zhong Guo, Hong-Yu Li, Ji-Jun Zho, Wen-D Xu, Deprtment of Gstroenterology, Shenyng Generl Hospitl of PLA, Shenyng , Lioning Province, Chin Author contributions: Chen J performed the mjority of experiments nd nlyzed the dt; Li HY nd Zho JJ performed the moleculr investigtions; Guo XZ designed nd coordinted the reserch; Chen J nd Xu WD wrote the pper. Supported by Ntionl Nturl Science Foundtion of Chin, No Institutionl review bord sttement: This study ws pproved by the Ethics Committee of Shenyng Generl Hospitl of PLA. Institutionl niml cre nd use committee sttement: There ws no niml experimenttion in this study. Conflict-of-interest sttement: The uthors hve no conflict of interests to declre. Dt shring sttement: No dditionl dt re vilble. Open-Access: This rticle is n open-ccess rticle which ws selected by n in-house editor nd fully peer-reviewed by externl reviewers. It is distributed in ccordnce with the Cretive Commons Attribution Non Commercil (CC BY-NC 4.0) license, which permits others to distribute, remix, dpt, build upon this work non-commercilly, nd license their derivtive works on different terms, provided the originl work is properly cited nd the use is non-commercil. See: licenses/by-nc/4.0/ Mnuscript source: Unsolicited mnuscript Correspondence to: Xio-Zhong Guo, MD, Deprtment of Gstroenterology, Shenyng Generl Hospitl of PLA, No. 83 Wenhu Rod, Shenyng , Lioning Province, Chin. guoxiozhong1962@liyun.com Telephone: Fx: Received: September 13, 2016 Peer-review strted: September 14, 2016 First decision: October 20, 2016 Revised: November 4, 2016 Accepted: December 21, 2016 Article in press: December 21, 2016 Published online: Februry 7, 2017 Abstrct AIM To investigte the enhnced cytotoxic T lymphocyte responses ginst pncretic cncer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete nti-dcr3 monoclonl ntibody (mab). METHODS DCs, T lymphocytes nd primry PC cells were obtined from PC ptients. DCs were trnsfected with designed humnized nti-dcr3 monoclonl ntibody hevy nd light chin mrna nd/or totl tumor RNA (DC-tumor-nti-DcR3 RNA or DC-totl tumor RNA) by using electroportion technology. The identifiction, concentrtion nd function of nti-dcr3 mab secreted by DC-tumor-nti-DcR3 RNA were determined by western blotting nd enzyme-linked immunosorbent ssy. After co-culturing of utologous isolted PC cells with trget DCs, the effects of secreting nti-dcr3 mab on RNA-DCs vibility nd poptosis were ssessed by MTT ssy nd flow cytometry. Anlysis of enhnced ntigen-specific immune response ginst PC induced by nti-dcr3 mab secreting DCs ws performed using 51 Cr relesing test. T cell responses induced by RNAloded DCs were nlyzed by mesuring cytokine levels, including IFN-γ, IL-10, IL4, TNF-α nd IL-12. RESULTS The nti-dcr3 mab secreted by DCs rected with 817 Februry 7, 2017 Volume 23 Issue 5

2 Chen J et l. CTL responses to PC induced by DCs recombinnt humn DcR3 protein nd generted bnd with 35 kd moleculr weight. The secreting mab ws trnsient, peking t 24 h nd becoming undetectble fter 72 h. After co-incubtion with DCtumor-nti-DcR3 RNA for designted times, the DcR3 level in the superntnt of utologous PC cells ws significntly down-regulted (P < 0.05). DCs secreting nti-dcr3 mab could improve cell vibility nd slow down the poptosis of RNA-loded DCs, compred with DC-totl tumor RNA (P < 0.01). The nti-dcr3 mab secreted by DC-tumor-nti-DcR3 RNA could enhnce the induction of cytotoxic T lymphocytes (CTLs) ctivity towrd RNA-trnsfected DCs, primry tumor cells, nd PC cell lines, compred with CTLs stimulted by DC-totl tumor RNA or control group (P < 0.05). Menwhile, the ntigen-specific CTL responses were MHC clss I-restricted. The CD4+ T cells nd CD8+ T cells incubted with nti-dcr3 mab secreting DCs could produce extremely higher level IFN-γ nd lower level IL4 thn those incubted with DC-totl tumor RNA or controls (P < 0.01). CONCLUSION DCs engineered to secrete nti-dcr3 ntibody cn ugment CTL responses ginst PC in vitro, nd the immune-enhncing effects my be prtly due to their cpbility of down-regulting DC poptosis nd djusting the Th1/Th2 cytokine network. Key words: Dendritic cell; Antibody-encoding RNA; DcR3; Cytotoxic T lymphocyte response; Pncretic cncer The Author(s) Published by Bishideng Publishing Group Inc. All rights reserved. Core tip: Dendritic cells co-trnsfection with tumorssocited ntigens RNA nd humnized nti-dcr3 monoclonl ntibody mrna my ugment cytotoxic T lymphocyte responses ginst pncretic cncer in vitro. This finding lys good foundtion for further investigtion of tumor dendritic cells vccine trgeting DcR3 protein ginst pncretic cncer. Chen J, Guo XZ, Li HY, Zho JJ, Xu WD. Dendritic cells engineered to secrete nti-dcr3 ntibody ugment cytotoxic T lymphocyte response ginst pncretic cncer in vitro. World J Gstroenterol 2017; 23(5): Avilble from: URL: DOI: dx.doi.org/ /wjg.v23.i5.817 INTRODUCTION Pncretic cncer (PC) is the fourth leding cuse of cncer-relted deths in the US, with deths in 2015 lone [1,2]. It hs n extremely poor prognosis with totl 5-yer survivl rte of < 5% [3]. The poor prognosis nd high mortlity rte in PC ptients my be ttributed in prt to lck of effective tretments [2]. Existing therpies for PC re limited to systemic chemotherpy nd surgicl resection. However, neither of these two strtegies cn cure PC completely [4]. Thus, more effective therpeutic methods re urgently needed. Cellulr immunotherpy is promising lterntive tht is currently considered the fourth line of cncer tretment [5]. In this pproch, different kinds of immune cells, such s cytokine-induced killer cells, lymphokinectivted killer cells, nturl killer (NK) cells nd dendritic cells (DCs) re dopted for immunotherpy. Of these, DC is the most commonly used immune effector cell becuse of its potent ntigen-presenting function in the initition of ntitumor immune responses nd its pivotl function in cncer immunosurveillnce. Cytotoxic T lymphocytes (CTLs) re cpble of eliminting cncer cells directly in vivo, but their ctivities re primrily mnged by DCs. The use of DC-bsed tumor vccines hs therefore become promising lterntive tretment method for cncer [2,6]. In clinicl prctice, ntigen choice is essentil in the design of n effective vccine. Becuse of lcking the expression of MHC clss II molecules nd co-stimultory molecules, PCs, with low level of expression of tumorssocited ntigens (TAA), disply wek ntigenicity nd high heterogeneity [7]. Therefore, loding whole ntigens from PC cells my be n lterntive method tht cn both generte brod T cell immune response to TAA nd reduce the possibility of PC escpe from immune recognition. We hve previously reported tht DCs trnsfected with totl tumor RNA cn effectively induce nti-pc tumor-specific CTL responses [2]. However, lthough this method hs been demonstrted to generte vccine-induced rise in tumor-specific cells, the immune nd tumor rections sty modest, suggesting the need for novel strtegies to improve ntitumor immunity [8]. Evidently, one cuse of this insufficiency is tht tumor cells cn produce certin immunosuppressive molecules to induce n immunosuppressive microenvironment nd inhibit the function of tumor-ssocited cells, such s T lymphocytes nd DCs [9]. Decoy receptor (DcR) 3 is possibly one of these cells in the tumor microenvironment (TME) [10]. DcR3 is decoy receptor for Fs lignd (FsL) nd is member of the tumor necrosis fctor receptor (TNFR) superfmily [11]. DcR3 displys inducible expression, intercts with the herpes virus entry meditor (HVEM) nd is presented by TNF-like molecule 1A (TL1A) nd T lymphocytes (LIGHT) [12,13]. DcR3 lcks trnsmembrne domin nd works s secreted protein insted of membrne-bound one. DcR3 cn bind to LIGHT nd FsL, thereby blocking the interction between LIGHT nd LT-receptor (LTbR) or HVEM to inhibit the poptosis induced by Fs-FsL interction or LIGHT-medited biologicl effects. DcR3 is frequently overexpressed in vrious 818 Februry 7, 2017 Volume 23 Issue 5

3 Chen J et l. CTL responses to PC induced by DCs tumors, including lung cncers [14], gstrointestinl trct tumors [15], virus-ssocited lymphoms [16], nd PCs [17]. It hs been reported tht overexpression of DcR3 is correlted with shortened totl survivl time of cncer ptients [18]. DcR3 hs been postulted to promote tumor growth by escping FsL- nd LIGHTmedited immunosurveillnce. Specificlly, DcR3 is ble to suppress the ctivtion nd differentition of DCs nd mcrophges [19], enhnce osteoclst differentition nd ngiogenesis [20], nd sensitize T lymphocytes to TNF-relted poptosis-inducing lignd (TRAIL)-induced poptosis in cncer ptients [21]. In ddition, DcR3 is lso regrded s n importnt immunosuppressive fctor in defects ssocited with immune effector cell function. Therefore, we sought to determine whether neutrlizing DcR3 expression in the TME cn ugment the CTL responses ginst PC in vitro induced by DCs loded with totl tumor RNA. In the current study, we evluted the novel pproch of co-trnsfecting DCs with totl tumor RNA nd mrna encoding humnized hevy (H) nd light (L) chins of n nti-humn DcR3 mab together to chieve nti-dcr3 protein stimultion. Through co-culturing of utologous isolted PC cells with DCs, we found tht DCs trnsfected with these RNAs secrete opertionl immune modulting proteins tht cn reduce DcR3 expression in TME of cultured PC cells. Then we demonstrted tht CTLs induced by DCs co-trnsfected with totl tumor RNA nd nti-dcr3 monoclonl ntibody (mab) mrna show more effective cytotoxic ctivities ginst PC cells in vitro compred with DCs loded only with totl tumor RNA lone. Furthermore, the immune-enhncing effect of DCs engineered to secrete nti-dcr3 mab is prtly due to their cpbility of down-regulting poptosis of DCs nd djusting the T helper (Th)1/Th2 cytokine network. These findings re crucil for the development of tumor DC vccines trgeting DcR3 protein ginst PC. MATERIALS AND METHODS Ptient eligibility nd tumor cells preprtion Fifteen HLA-A2+ PC ptients (9 mles nd 6 femles; medin ge of 53.5 yers, rnging from 35 yers to 72 yers) were included in this study. According to the TNM clssifiction of AJCC [22], there were 10 stge II ptients nd 5 stge Ⅲ ptients. The loction of tumor ws divided into hed (7 cses) nd body/til (8 cses). All ptients underwent surgicl resection nd were pthologiclly dignosed with invsive ducl denocrcinom. Peripherl blood monocyte cells (PBMCs), isolted by Ficoll-Hypque (Sigm, St Louis, MO, United Sttes) density grdient seprtion, nd ws used s the nonmlignnt control tissues. Pncretic cncer specimens were obtined t the time of surgery nd were stored in RNAlte (Ambion, Austin, TX, United Sttes) t 4 until processing. Autologous tumor cells were obtined s described by Wng et l [23]. Approximtely 10 g of ech tumor specimen ws hrvested in the operting room for primry cell culture. The tumor tissue ws mechniclly disrupted to generte pproximtely 1 mm 3 sections. The tissue ws digested in 10 ml of RPMI-1640 medium supplemented with 0.05% collgense (Hyclone, South Logn, UT, United Sttes) with gentle gittion t room temperture for 4-6 h. After culturing for 7 d, the immunohistochemistry technique ws used to detect the expression of DcR3 protein (nti-dcr3 mab obtined from Sigm). The humn PC cell lines Cpn-2 (HLA-A2+) nd AsPC-1 (HLA-A2-), s well s the leukemi cell line K562, were obtined from the Americn Type Culture Collection (Mnsss, VA, United Sttes). The cells were cultured in RPMI-1640 supplemented with 10% fetl bovine serum, 2 mm L-glutmine (Hyclone), 50 U/mL penicillin, nd 50 mg/ml streptomycin (Hyclone). All cells were cultured for 7 d nd mintined in the logrithmic phse growth t 37 in humidified tmosphere supplemented with 5% CO2. Preprtion of RNA Totl cellulr RNA ws extrcted from utologous PC cells nd PBMCs by using TRIzol Regent (Sigm) ccording to the mnufcturer s instructions. Only RNA exhibiting rtio of 28S:18S > 1 ws subjected to further nlysis. Totl RNA of nti-humn DcR3 hybridom clone 1B1 ( kindly gift from Dr. CF Wu of Jilin University, Chin) [24], ws isolted with the RNesy mini kit (Qigen, Vlenci, CA, United Sttes). Five microgrms of RNA were used in reverse trnscription rection with the RT primer 5 - ATT CTA GAG GCC GAG GCG GCC GAC ATG (T-30) VN-3 nd PowerScript RT (Clontech, Mountin View, CA, United Sttes) nd the primer 5 -AAG CAG TGG TAT CAA CGC AGA GTG GCC ATA TTG GCCr GrGrG 3AmMC7/-3. The hevy (H) chin ws mplified from 10% of the RT rection by using Advntge 2 HF PCR mix (Clontech) nd the primers 5 - AAA GAA TTC GGC CTT GTT GGC CTC ATT TAC CCA GAG ACC GGG AGA TG -3 nd 5 -GAA AAG CTT GGC CAT TGG GGC GGT ATC AAC GCA GAG TGG CCA TAT TG-3. The L chin ws mplified with the primers 5 -AAG AAT TCG GCC TTG TTG GCC TAA CAC TCA TTC CTG TTG AAG CTC TTG-3 nd 5 -GAA AAG CTT GGC CAT TGG GGC GGT ATC AAC GCA GAG TGG CCA TAT TG-3. The resulting PCR frgments were digested with HindⅢ nd EcoRI nd cloned into the HindⅢ nd EcoRI sites of the plsmid psp73-sph/a64, which possesses T7 promoter nd 64T nucleotides tht llow for the production of in vitro trnscribed RNA with polya til of 64 residues. The gene encoding the full-length enhnced ctin (s controls) ws inserted into the psp73-sph/a64 plsmid, s well. In vitro trnscription of mrna All plsmids were digested with SpeI for use s templte for in vitro trnscription rections using the 819 Februry 7, 2017 Volume 23 Issue 5

4 Chen J et l. CTL responses to PC induced by DCs mmessage mmachine T7 kit (Ambion) ccording to the mnufcturer s protocol. mrna ws purified with the RNesy mini kit. Genertion nd electroportion of DCs DCs genertion ws performed s previously described by Zhu et l [25]. A concentrted leukocyte frction ws isolted from PBMCs tht processed 200 ml of blood during ech collection. Leukpheresis products were seprted by density-grdient centrifugtion over polysucrose sodium ditrizote (Sigm), nd cells were resuspended in serum-free AIM-V medium (Gibco, Burlington, Cnd). Cells were incubted in humidified incubtor for 2 h t 37 to llow plstic dherence. The non-dherent frction ws removed, nd the dherent cells were cultured for 7 d in serumfree AIM-V medium supplemented with humn ril-4 (500 U/mL) nd recombinnt humn grnulocyte mcrophge colony-stimulting fctor (GM-CSF) (800 U/mL) (R&D Systems, Minnepolis, MN, United Sttes) t 37 under 5% CO2. The DCs were trnsfected with RNA using Gene Pulser II (Bio-Rd, Hercules, CA, United Sttes). After 7 d, the immture DCs were trnsferred into lowconductnce medium (Cytofusion Medium Formul C; CytoPulse Sciences, Columbi, MO, United Sttes) fter centrifugtion t 170 g t 4 for three times, ech time for 7 min. Vible cells were resuspended to finl concentrtion of (10-40) 10 6 cells/ml in the low-conductnce medium. Subsequently, 0.5 ml cell suspension ws mixed with 3 μg per 10 6 DCs nd electroported in 0.4 cm cuvette t n optimum condition [26]. The cells were recovered for 5 min, nd then the sme protocols were repeted with 10 μg of H chin ntibody RNA nd 5 μg of L chin ntibody RNA per 10 6 DCs. Cells were recovered for 15 min, nd then the trnsfected DCs were mtured by dding 10 ng/ml of TNF-α (Roche Moleculr Biochemicls, Mnnheim, Germny) for 24 h. The electroportion of ctin mrna nd PBMC RNA ws used s controls. Inverted phse contrst microscopy (I 70; Olympus, Tokyo, Jpn) nd electron microscopy (scnning electron microscope JSM-7300EX; Hitchi, Tokyo, Jpn) were used for the morphologicl chrcteriztion of DCs trnsfected with different RNA. The DcR3 protein expression in RNA- DCs ws low to negtive (mture DC 11%-14% nd immture DC 9%-11%, dt not shown). Western blot nlysis The recombinnt humn DcR3 protein (Sigm) ws dissolved in 0.02 mol/l phosphte-buffered sline (PBS, ph 7.4). The concentrtion ws determined by the BCA Protein Assy Regent method (Pierce Chemicl Compny, Rockford, IL, United Sttes). Then, the proteins were resolved on sodium dodecyl sulfte (SDS)-polycrylmide denturing gels, nd trnsferred onto nitrocellulose membrnes (Schleicher nd Schuell, Dssel, Germny) overnight t 4. The membrnes were blocked in Tris-buffered sline contining 2% non-ft dry milk (Bio-Rd) nd 0.05% Tween 20 (Sigm) for 1 h. Using 1:5000 dilution of the superntnt from DCs co-trnsfected with utologous PC cell totl RNA nd nti-dcr3 mab-encoding mrna for 24 h, the superntnt from DCs ws trnsfected with ctin mrna for 24 h (negtive control) nd the commercil nti-dcr3 mab (positive control; Qigen) s primry ntibodies, followed by incubtion for 1 h in horserdish peroxidse-conjugted secondry ntibody. Protein bnds were visulized using n enhnced chemiluminescence system (Amershm Phrmci Biotech, Pisctwy, NJ, United Sttes). Enzyme-linked immunosorbent ssy As described by our previous study [26], utologous tumor cells ( cells) were co-incubted with DCs ( cells) encoding nti-dcr3 mab mrna or ctin mrna (negtive control) in 96-well pltes in n overll volume of 200 μl t 37 for 0-72 h. A 1:5000 dilution of the commercil nti-dcr3 mab ws pplied s positive control. Triplicte superntnt smples from these co-cultures were exmined for specific DcR3 secretion using DcR3 enzyme-linked immunosorbent ssy (ELISA) kit (Genzyme, Wlthm, MA, United Sttes). Ech well ws mesured t 450 nm, nd the opticl density vlues were used to clculte the concentrtion of the smples. As previously described by Pruitt [8], n indirect ELISA ws simultneously used to mesure the DcR3 mab concentrtion in the superntnts of DCs cotrnsfected with totl tumor RNA nd nti-dcr3 mabencoding mrna for 0-72 h. DCs trnsfected with ctin mrna were used s positive control. Assy for DCs vibility As described by Chen [27], the 3-(4,5-dimethylthizol- 2-yl)-2,5-diphenyl-2H-tetrzolium bromide (MTT) cell prolifertion ssy ws used to defined the cell vibility. In brief, RNA loded DCs were inoculted into 96-well tissue culture pltes (BD PhrMingen, Sn Diego, CA, United Sttes) with density of 3000 cells per well nd co-incubtion with or without 3000 utologous PC cells. The cells were hndled ccording to the instructions of MTT t the time points of 0-96 h. The formzn crystls tht we cquired were dissolved in dimethylsulfoxide (BD Biosciences, Frnklin Lkes, NJ, United Sttes). Absorbnce ws monitored t 490 nm nd the percentge of exposed cells to controls ws used to show the cell vibility. Cells tht did not receive RNA trnsfections were regrded s the control cultures. Phenotypic nlysis of DCs by flow cytometry As described by our previous study [2], DCs were prepred using 2% prformldehyde fter wshing for three times with frigid PBS contining 0.5% of bovine serum lbumin. Four fluorescein isothiocynte 820 Februry 7, 2017 Volume 23 Issue 5

5 Chen J et l. CTL responses to PC induced by DCs (FITC)-conjugted mabs, including nti-hla-dr, nti- CD80, nti-cd86 nd nti-cd83, were used nd which cme from BD PhrMingen. Flow cytometry ws used to nlyze those stined cells. Flow cytometric nlysis of DC poptosis Following the mnner of Lin et l [28], poptotic DCs were quntified by using nnexin V-FITC nd propidium iodide (PI) double stining. Briefly, utologous tumor cells were co-incubted with DC-totl tumor RNA nd DC-tumor-nti-DcR3 mab mrna for the specific times, nd then cells were resuspended in 100 μl binding buffer, fter wshing with frigid PBS. Two microliters of PI nd FITC-nnexin V were dded into the cells tht were resuspended nd cultured for 15 min protecting from light. The cells were then dded into 0.5 ml binding buffer nd nlyzed with flow cytometry. Induction of ntigen-specific CTL nd in vitro cytotoxicity ssy Antigen-specific CTLs were produced utilizing protocol described by Chen et l [2,26,27]. The PBMCs without dherence were cultured in serum-free medium contining 10 ng/ml IL-7 nd 20 U/mL IL-2 (R&D Systems). The cells were encourged weekly for minimum of two times with RNA-DCs t stimultorto-effector rtio of 1:10. As determined by flow cytometric nlysis, minimum of 45% of purified effector cells were CD8+ T cells fter 16 d of culture. Trget cells, including utologous DCs trnsfected with tumor ntigen-encoding RNA nd tumor cells, were resuspended in 1 ml RPMI-1640 medium t 37 in 5% CO2 for 1 h, nd which contined 100 μci NCrO4 solution (Isotope Products, Beijing, Chin). The seril dilutions of effector CTLs t vrious E:T rtios nd the 5 51 Cr-lbeled trget cells were incubted in 200 μl RPMI-1640 in 96-well pltes for 6 h. Fifty microliters of superntnt were then tken wy, nd 51 Cr secretion ws mesured by gmm counter (Beckmnn, Heidelberg, Germny). In ll the tests, the spontneous dischrge ws less thn 15% of the totl relese of the detergent. Specific lysis percentge ws computed s [(experimentl cpm-spontneous cpm)/(mximum cpm-spontneous cpm)] 100. Anlysis of cytokines relesed by T cells As described by our previous study [2], fter subjecting to different tretments, 5 DCs (DC-totl tumor RNA nd DC-tumor-nti-DcR3 RNA) were cultured in 96-well round bottom pltes. T cells were isolted from proliferting peripherl blood lymphocytes (PBLCs) nd 5 T cells were stimulted with RNA-DCs in whole volume of 200 μl in 96-well pltes for 24 h. The cytokines interleukin (IL)-12p70, interferon-γ (IFN-γ), IL-10, nd TNF-α relesed by T cells were mesured by ELISA kits (Endogen, Woburn, MA, United Sttes). The results were obtined from triplicte wells nd the exmintion of superntnt from cultured T cells lone for the four cytokines were used s control groups. RNA-loded DC-induced CD4+ nd CD8+ T cell responses The mesurement of multi-ntigen specific CD4+ nd CD8+ T cell responses were conducted by cytokine relese ssy s described by Chen et l [2] nd Miyzw et l [29]. Through using the instrument of utomacs TM (Miltenyi Biotec, Bergisch Gldbch, Germny), CD4+ nd CD8+ T cells were seprted from proliferting PBLCs nd tht were civilized fter three cycles of re-stimultion ex vivo. The cells were then incubted with CD4 or CD8 microbeds (Miltenyi Biotec) for 15 min t 4 nd wshed before seprtion. Seprtion ws executed dopting n utomacs column (Miltenyi Biotec). The pillr ws set in the mgnetic field, nd mgneticlly-lbeled cells were preserved in the pillr nd then flushed out s positively chosen cells while the mgnetic field ws turned off. The sorted popultions purity ws determined by flow cytometry. The selected CD4+ nd CD8+ T cells (5 ) were stimulted with RNA- DCs (DC-ctin mrna, DC-PBMC RNA, DC-totl tumor RNA, nd DC-tumor-nti-DcR3 mab RNA, 5 ) in n overll volume of 200 μl of the entire medium in 96-well pltes for 24 h. The superntnts were collected, nd the IL-4 nd IFN-γ levels were mesured using IL-4 nd humn IFN-γ ELISA kits (Endogen). Ech ssy ws crried out on duplicte smples. Sttisticl nlysis Quntittive dt re presented s men ± SD. The ANOVA nd post hoc test (S-N-K method) nlyses were performed using Excel softwre (Microsoft, Redmond, WA, United Sttes). P < 0.05 ws considered for sttisticl significnce. RESULTS Genertion of primry tumor cells nd DCs Primry tumor cells nd DCs were cultured from HLA-A2+ PC ptients. Most cultured primry tumor cells (> 90%) were found to be positive for DcR3 in the cytoplsm (Figure 1A). For DCs, cells cultured from PBMCs with stimultion of GM-CSF, TNF-α nd IL-4 showed series of typicl morphologies of mture DCs. Most DCs without trnsfection with RNAs ssembled non-cohesive colonies. Cells tht were blted from these colonies demonstrted typicl villiform processes s shown by inverted phse contrst microscopy (Figure 1B). Both trnsfection with totl tumor RNA (Figure 1C) nd simultneously loding with the totl tumor RNA nd nti-dcr3 mab mrna (Figure 1D) showed typicl morphologicl chrcteristics of DCs. DC-tumor-nti-DcR3 RNA secrete functionl nti-dcr3 mab To determine if we could produce specific mab by 821 Februry 7, 2017 Volume 23 Issue 5

6 Chen J et l. CTL responses to PC induced by DCs A B C D Figure 1 Cells obtined represented typicl morphologicl chrcteristic of primry tumor cells nd mture dendritic cells. Cells were derived from five pncretic cncer (PC) ptients nd with similr shpe of trget cells. A: Most of the cultured primry tumor cells (over 90%) showed DcR3-positive expression (mgnifiction 200); B: After cultured for 7 d, mtured dendritic cells (DCs) were trnsfected without RNA nd ssembled into non-cohesive colonies, nd the blted cell showed distinctive villiform process (mgnifiction 200). DCs trnsfected with totl tumor RNA lone (C) or together with nti-dcr3 mab mrna (D) showed similr cell morphology to DCs trnsfected without RNA (C: mgnifiction 400; D: scnning electron microscope, br represents 10 μm). pulsing DCs with nti-dcr3 mab-encoding RNA, we electroported tumor-ntigens-loded DCs with IVT RNA encoding H nd L mab chins (nti-dcr3 H+L mrna). The superntnts of DCs were obtined t the designted time points to identify the trget mab nd mesure their concentrtion. The superntnts of cocultured utologous PC tumor cells nd DC-tumor-nti- DcR3 RNA were hrvested t specific time points to determine the effects of the mab (Figure 2). First, we used Western blotting to identify the nti- DcR3 mab produced by DCs co-trnsfected with totl tumor RNA nd nti-dcr3 mab mrna. As shown in Figure 2A, the superntnt of DC-tumor-nti-DcR3 RNA could specificlly neutrlize the recombinnt humn DcR3 protein nd generte bnd with slightly higher moleculr weight thn 30 kd, which ws in line with the theoreticl moleculr weight of DcR3 protein. A commercilly vilble nti-dcr3 mab ws used s positive control, while DC-ctin ws used s negtive control. The mounts of nti-dcr3 mab secreted by RNApulsed DCs were nlyzed using n indirect ELISA ssy. As shown in Figure 2B, mab production by DCtumor-nti-DcR3 RNA ws trnsient nd peked t 24 h (contining ± 1.9 ng of nti-dcr3 mab per cells) nd then could not be detected fter 72 h. However, no nti-dcr3 mab ws found in the superntnt of DC-ctin RNA t ny point. The specific ntigen-binding effect of nti-dcr3 mab secreted by DCs co-trnsfected with totl tumor RNA nd humnized nti-dcr3 mab mrna ws confirmed by ELISA. As shown in Figure 2C, the soluble DcR3 protein in the superntnt of utologous PC cells ( cells) co-cultured with DC-tumor-nti- DcR3 RNA ( cells) ws significntly lower thn tht of tumor cells nd the DC-ctin RNA co-incubtion group from 12 h to 72 h ( P < 0.05). Enhnced tumor-specific immune response induced by nti-dcr3 mab secreting DCs We next sought to determine whether nti-tumor responses could be enhnced by immunizing DCs cotrnsfected with totl tumor RNA nd nti-dcr3 mab mrna. Using cells from HLA-A2+ PC ptients, we evluted the bility of DC-tumor-nti-DcR3 RNA to ugment the induction of nti-pc CTLs in response to DCs. As shown in the left pnel of Figure 3A, DCtumor-nti-DcR3 RNA ws used s not only stimultor cells but lso trget cells, while DCs trnsfected with totl tumor RNA lone or other utologous RNA- DCs were used s trgets. DC-tumor-nti-DcR3 RNA demonstrted further enhncement of ntigen-specific CTL induction compred with DCs only loded with totl tumor RNA without ny increse in CTL ctivity bove non-specific bckground. CTL ctivity ginst PC cells ws lso ssessed. 822 Februry 7, 2017 Volume 23 Issue 5

7 Chen J et l. CTL responses to PC induced by DCs A B Anti-DcR3 mab (ng/ml) C DcR3 expression (ng/ml) kd DC-tumor-nti-DcR3 RNA DC-ctin mrna Times fter trnsfection (h) DC-ctin mrna DC-tumor-nti-DcR3 RNA Times fter DCs nd tumor cells coincubtion (h) Figure 2 Identifiction, concentrtion nd function of nti-dcr3 monoclonl ntibody secreted by dendritic cells co-trnsfected with totl tumor RNA nd nti-dcr3 monoclonl ntibody mrna. A: Western blotting nlysis showed tht, similr to commercil nti-dcr3 mab (lne 2), mab secreted by dendritic cells (DCs) co-trnsfected with totl tumor RNA nd humnized nti- DcR3 H+L mrna (lne 4) could lso rect with the recombinnt humn DcR3 protein (moleculr weight of 35 kd) nd generte bnd with moleculr weight slightly greter thn 30 kd, wheres the superntnt hrvested from DC-ctin RNA could not bind the DcR3 protein (lne 3); B: The mounts of nti- DcR3 mab produced by RNA trnsfected DCs were nlyzed using indirect ELISA ssy. The mab secreted by DC-tumor-nti-DcR3 RNA ws trnsient, peked t 24 h, nd then could not be detected fter 72 h. However, no nti- DcR3 mab ws found in the superntnt of DC-ctin RNA continuously; C: The specific ntigen binding effect of nti-dcr3 mab secreted by RNA trnsfected DCs ws determined by mesuring the levels of DcR3 protein in the superntnt of utologous tumor cells ( cells) co-cultured with defined DCs ( cells). After co-incubtion with DC-tumor-nti-DcR3 RNA for h, the soluble DcR3 protein level in the superntnt of utologous PC cells ws significntly lower thn those of tumor cells nd the DC-ctin RNA cocultured group ( P < 0.05). Except for those of western blotting nd histogrm, dt represent the mens of three experiments, nd the histogrms re representtive of three experiments. Error brs represent SD. mab: Monoclonl ntibody. Both CTLs induced by DC-tumor-nti-DcR3 RNA nd DC-totl tumor RNA were ble to lyse their own cncer cells effectively, while CTLs induced by DC-PBMC RNA or DC-ctin RNA were not, s shown in Figure 3A (right pnel). Furthermore, DC-tumor-nti-DcR3 RNA showed greter effectiveness nd superior bility to recognize nd lyse HLA-A2+ utologous PC cells (P < 0.05). Menwhile, with increse in the E:T rtio (from 10:1 to 40:1), the killing intensity incresed concomitntly (P < 0.05). No lysis of norml PBMCs or NK-sensitive K562 cells ws observed. However, evident lysis ginst the cultured PC cell line occurred (Figure 3B). Effector T cells (HLA-A2+) stimulted by DCs trnsfected with totl tumor RNA lone or together with nti-dcr3 mab mrna could lyse the Cpn-2 cells, which expressed the HLA-A2+ ntigen endogenously. On the other hnd, HLA-A2- AsPC-1 cells were not identified nd lysed. DC-tumor-nti-DcR3 RNA ws more potent t inducing cytotoxicity in CTLs ginst Cpn-2 cells with HLA-A2 dpttion in comprison with the CTLs induced by DC-totl tumor RNA lone (P < 0.05). The expression of HLA lleles other thn A2 ws not evluted in these experiments. Improvement in cell vibility by nti-dcr3 mab secreting DCs The effect of nti-dcr3 mab secreting DCs on vibility of RNA-loded DCs ws determined by MTT ssy. As shown in Figure 4A, when RNA-DCs were co-incubted without utologous tumor cells, cell vibility did not vry significntly t designed times, with round 85% survivl throughout (P > 0.05). By contrst, the vibility of DC-totl tumor RNA cultured with tumor cells ws evidently lower thn tht of DC-tumor-nti- DcR3 RNA (P < 0.01). The results of DC-PBMC RNA nd DC-ctin RNA were similr to those of DC-totl tumor RNA. Menwhile, both the DC-tumor-nti- DcR3 RNA nd DC-tumor RNA demonstrted positive expression of CD80, CD83, CD86 (co-stimultory molecules) nd HLA-DR (MHC II molecules) fter coincubtion with PC cells for 24 h, but there ws no significnt difference between the two methodologies (dt not shown). Annexin V nd PI double stining ws performed to demonstrte the inhibition of poptosis of DCs cotrnsfected with totl tumor RNA nd nti-dcr3 mab mrna. After incubtion with utologous PC cells for 0-96 h, nnexin V-positive poptotic cells were found to increse shrply in time-dependent mnner in DC-totl tumor RNA, wheres poptosis of DC-tumor-nti-DcR3 RNA were mitigted by nti-dcr3 mab (Figure 4B). T cell cytokine production pulsed by RNA-DCs There were no significnt differences in the mount of IL-10, TNF-α, IL-12p70 nd IFN-γ cytokines relesed in the culture superntnts of T cells when mesured by ELISA (dt not shown). However, high levels of cytokines could be secreted by T cells fter pulsing 823 Februry 7, 2017 Volume 23 Issue 5

8 Chen J et l. CTL responses to PC induced by DCs A 50 Stimultor: DC-tumor-nti-DcR3 RNA B Trget cells: Autologous pncretic cncer cells b 80 Cytotoxicity (%) Cytotoxicity (%) b DC-totl tumor RNA DC-PBMC RNA DC-ctin RNA DC-tumor-nti-DcR3 RNA 0 5:1 10:1 20:1 40:1 E:T rtio 0 5:1 10:1 20:1 40:1 E:T rtio C 80 Trget cells: K562/PBMC/Cpn-2/AsPc-1 Cytotoxicity (%) DC-totl tumor RNA DC-tumor-nti-DcR3 RNA 0 K562 PBMC Cpn-2 AsPc-1 Figure 3 Antigen-specific immune response ginst pncretic cncer is enhnced by nti-dcr3 monoclonl ntibody secreting dendritic cell. Dendritic cells (DCs)-totl tumor RNA, DC-PBMC RNA, DC-ctin RNA nd DC-tumor-nti-DcR3 RNA were used to stimulte utologous T cells weekly for two times followed by cytotoxic T lymphocyte (CTL) ssy. Induction of tumor ntigen-specific CTLs ws mesured by using RNA-trnsfected DCs nd tumor trgets (primry tumor cells, K562, Cpn-2 nd AsPC-1 cell line cells). A: Left pnel: DC-tumor-nti-DcR3 RNA ws used s not only stimultor cells but lso trget cells, nd CTLs stimulted by DC-tumor-nti-DcR3 RNA could recognize nd lyse tumor ntigen-specific cncer trgets (DC-totl tumor RNA nd DC-tumor-nti-DcR3 RNA). No cross-rectivity ws pprent ginst DCs loded with norml tissue surrounding PC or ctin (DC-PBMC RNA nd DC-ctin RNA). Compred with CTLs stimulted by DC-totl tumor RNA, DC-tumor-nti-DcR3 RNA further enhnced the induction of CTL ctivity. Right pnel: Both DCs co-trnsfected with totl tumor RNA nd nti-dcr3 mab mrna nd DCs trnsfected with totl tumor RNA lone showed n effective nd superior bility in recognizing nd lysing HLA-A2+ utologous PC cells, wheres T cells ctivted by DC-PBMC RNA or DC-ctin RNA could not ( P < 0.05). Moreover, CTLs induced by DC-tumor-nti-DcR3 RNA could produce more powerful killing ctivity towrd the tumor cells compred with the DC-totl tumor RNA with the E:T rtio incresing from 10:1 to 40:1 ( b P < 0.05); B: At the E:T rtio of 40:1, the effector T cells (HLA-A2+) stimulted by DCs trnsfected with totl tumor RNA lone or together with nti-dcr3 mab mrna could lyse the Cpn-2 cell line cells, which endogenously expressed the HLA-A2 ntigen effectively. By contrst, the cells of the AsPC-1 line (HLA-A2-) were not recognized nd lysed. DC-tumor-nti-DcR3 RNA showed more powerful cpbility in inducing the cytotoxicity of CTLs ginst HLA-A2-mtched tumor cell line (Cpn-2) compred with tht induced by DC-totl tumor RNA lone ( P < 0.05). The experiment ws repeted thrice representtively, nd the dt re shown s men ± SD. mab: Monoclonl ntibody; DC: Dendritic cell; CTLs: Cytotoxic T lymphocytes. with RNA-DC (Figure 5A). The IFN-γ nd IL-12p70 produced by DC-tumor-nti-DcR3 RNA pulsed T cells were higher thn those secreted by T cell pulsed by DC-totl tumor RNA (P < 0.01). At the sme time, compred with the DC-totl tumor RNA group, the TNF-α levels detected in the DC-tumor-nti-DcR3 RNA group ws not chnged significntly (P > 0.05). In ddition, the IL-10 level ws lower when it ws detected in DCs co-trnsfected with both totl tumor RNA nd nti-dcr3 mab RNA (P < 0.05). CD4+ nd CD8+ T cell responses induced by nti-dcr3 mab secreting DCs As shown in Figure 5B, the CD4+ nd CD8+ T cells incubted with DCs trnsfected with totl tumor RNA lone or together with nti-dcr3 mab mrna produced significntly higher level of IFN-γ thn those incubted with control DC (DC-ctin RNA) or DCs exposed to norml tissues (DC-PBMC RNA) (P < 0.01). In ddition, the CD4+ nd CD8+ T cells tht were cultured with DCs co-trnsfected with both totl tumor RNA nd nti-dcr3 mab RNA produced higher level of IFN-γ thn those cultured with DCtotl tumor RNA (P < 0.01). Menwhile, CD4+ nd CD8+ T cells incubted with DCs trnsfected with totl tumor RNA lone or together with nti-dcr3 mab mrna were ble to produce IL-4, but negtive or wek secretion of IL-4 ws observed in control groups (DC-ctin RNA nd DC-PBMC RNA) (P < 0.01). Furthermore, decresed levels of IL-4 production were detected in CD4+ T cells stimulted by DC-tumornti-DcR3 RNA compred with DC-totl tumor RNA (P < 0.01). No significnt difference in IL-4 secretion ws detected between CD8+ T cells stimulted by DC-tumor RNA nd those stimulted by DC-tumornti-DcR3 RNA. 824 Februry 7, 2017 Volume 23 Issue 5

9 Chen J et l. CTL responses to PC induced by DCs A Cell vibility (% of control) b DC-tumor-nti-DcR3 RNA DC-totl tumor RNA DC-tumor-nti-DcR3 RNA cultured with tumor cells DC-totl tumor RNA cultured with tumor cells Times fter trnsfection (h) B PI 0 h 24 h 48 h 72 h 96 h (times fter trnsfection) 0.62% 5.02% 9.65% 15.14% 25.25% DC-totl tumor RNA cultured with tumor cells 1.73% 1.87% 3.11% 8.12% 12.06% 0.87% 1.14% 3.93% 7.63% 12.98% DC-totl-nti-DcR3 RNA cultured with tumor cells 1.86% 2.07% 2.30% 2.84% 7.88% Annexin V Figure 4 Effects of nti-dcr3 monoclonl ntibody secreting dendritic cells on cell vibility nd poptosis in tumor RNA-loded dendritic cells. A: Dendritic cells (DCs) secreting nti-dcr3 mab improved the vibility of RNA-loded DCs. The vibilities of DC-totl tumor RNA nd DC-tumor-nti-DcR3 RNA, cultured with or without utologous tumor cells, were mesured using MTT ssy fter trnsfection for 0-96 h. The vibility of DCs trnsfected with totl tumor RNA lone or together with nti-dcr3 mab mrna did not chnge significntly t the designed time points, with pproximtely 85% survivl throughout ( P > 0.05). Within the sme period, the vibility of DC-totl tumor RNA cultured with tumor cells ws evidently lower thn tht of DC-tumor-nti-DcR3 RNA nd decresed in time-dependent mnner ( b P < 0.01). Three representtive experiments were run, nd the dt re shown s men ± SD; B: DC-totl tumor RNA nd DC-tumor-nti-DcR3 RNA were co-cultured with utologous pncretic cncer (PC) cells ( ) for 0, 24, 48, 72 nd 96 h. Cells stined with nnexin V-FITC nd propidium iodide were nlyzed by flow cytometry. The percentge of nnexin V-positive poptotic cells in DCs trnsfected with totl tumor RNA mrkedly incresed in time-dependent mnner, wheres the poptotic cells incresed slowly in DCs co-trnsfected with totl tumor RNA nd nti-dcr3 mab mrna. mab: Monoclonl ntibody. DISCUSSION Tumor cells cn produce severl immunomodultory molecules to induce immunosuppressive TME nd inhibit the function of tumor-ssocited DCs [9,30]. Therefore, new DC-bsed strtegies for producing tumor vccines re necessry to brogte immunosuppressive molecules in tumor tissue-induced mechnisms for suppressing the ctivtion of CTL responses tht cn tret estblished cncers [8]. DcR3 is one of the cndidte trget tumor-derived fctors [31]. As we initilly demonstrted, most cultured primry tumor cells showed DcR3-positive expression in the cytoplsm of PC ptients, which is consistent with the findings of Zhou et l [17]. Tumor cells engineered to relese high mounts of DcR3 re ble to protect themselves from poptosis, consequently resulting in decresed immune response nd suggesting tht DcR3 is involved in the immune evsion of mlignnt tumors [19,32]. As powerful immunomodultory fctor, DcR3 cn suppress ctin polymeriztion in mitogen-stimulted T cells, prevent the formtion of pseudopodi, down-regulte the ctivtion of DCs nd mcrophges, induce bnorml ggregtion of T cells fter ntigen stimultion, reduce the interction between T cells nd DCs, inhibit T cell chemotxis, nd induce T cell poptosis [19,33]. Therefore, neutrlizing the DcR3 protein secreted by tumor cells is prticulrly importnt in cncer immunotherpy. Different methods hve been developed for neutrlizing immunosuppressive fctors relesed by tumor cells. The effects of systemic dministrtion of ntibodies hve been exmined by numerous studies. Their findings showed tht mabs cn improve specil immune responses when dministered systemiclly [34]. Humn studies, however, hve reveled tht the side-effects of mabs re unvoidble when delivered systemiclly, nd one mjor concern is induction of utoimmunity [35-37]. Thus, our present study shows n lterntive strtegy of delivering mab by trnsfecting DCs with the RNA tht encodes both PC tumorntigens nd defined immunosuppressive molecule, nmely humnized nti-dcr3 mab. Using this strtegy, DCs were used both to produce nti-dcr3 mab nd 825 Februry 7, 2017 Volume 23 Issue 5

10 Chen J et l. CTL responses to PC induced by DCs A 400 DC-tumor-nti-DcR3 RNA DC-totl tumor RNA B DC-ctin RNA CD8 + T cell CD4 + T cell pg/ml DC-totl tumor RNA DC-tumor-nti-DcR3 RNA, b, b DC-PBMC RNA 0 IFN-g IL-12p70 IL-10 TNF IFN-g (pg/ml per 24 h) C DC-ctin RNA CD8 + T cell CD4 + T cell DC-totl tumor RNA, b DC-tumor-nti-DcR3 RNA, b DC-PBMC RNA IL4 (pg/ml per 24 h) Figure 5 T cell responses induced by nti-dcr3 monoclonl ntibody secreting dendritic cells. The T cells were co-cultured with RNA-dendritic cells (DCs) for 24 h. Then, the superntnts were collected, nd the cytokine levels were mesured by ELISA ssy. A: ELISA test showed the cytokines of IL-12p70 nd IFN-γ secreted by T cells pulsed by DC-tumor-nti-DcR3 RNA were higher thn those secreted by T cells pulsed by DC-totl tumor RNA without significnt chnge of TNF-α. Menwhile, IL-10 level ws lower in DC-tumor-nti-DcR3 RNA thn in DC-totl tumor RNA ( P < 0.05); B: CD4 + T cells nd CD8 + T cells incubted with DCs encoding whole tumor ntigens could produce extremely higher IFN-γ levels compred with those incubted with DCs s control or DCs treted with norml tissues ( P < 0.01). Furthermore, the CD4 + T nd CD8 + T cells incubted with DCs co-trnsfected totl tumor RNA nd nti-dcr3 mab mrna produced higher IFN-γ levels thn those incubted with DC-totl tumor RNA ( b P < 0.01); C: The CD4 + T nd CD8 + T cells incubted with DCs loded with whole tumor ntigens showed positive expression of IL-4, wheres negtive or wek expression of IL-4 ws observed in DC-ctin RNA nd DC-PBMC RNA cells ( P < 0.01). Moreover, decresed levels of IL-4 production were detected in CD4 + T cells stimulted by DCs co-trnsfected with totl tumor RNA nd nti-dcr3 mab mrna compred with DCs trnsfected with totl tumor RNA individully ( b P < 0.01). The experiment ws repeted three times representtively, nd results re shown s men ± SD. IL: Interleukin; ELISA: Enzyme-linked immunosorbent ssy; PBMC: Peripherl blood monocyte cell; mab: Monoclonl ntibody. s vehicles for delivery to the site of T cell ctivtion considering their well-documented function s ntigenpresenting cells. Our results showed tht the superntnt of cultured DCs co-trnsfected with totl tumor RNA nd nti-dcr3 H+L mrna could specificlly bind the recombinnt humn DcR3 protein nd generte bnd with moleculr weight tht ws slightly greter thn 30 kd, which ws in line with the theoreticl moleculr weight of DcR3 protein. In ddition, the DcR3 level in the superntnt of utologous PC cells co-cultured with DC-tumor-nti-DcR3 RNA ws significntly lower thn tht of the control group. These dt suggest tht this pproch is fesible nd cn generte sufficient nti- DcR3 mab to neutrlize the DcR3 secreted by PC cells in vitro. One dvntge of the locl relese of nti-dcr3 mab tht is provided vi DC mrna trnsfection is its secretion over reltively short time spn t the precise site where T cell ctivtion is needed [8]. We previously showed tht mrna subunits hve brief hlf-life of less thn 24 h fter DC trnsfection [2], implying tht mrna trnslting into protein is n instntneous event in DC. Here, we confirmed tht for nti-dcr3 mab, most trget proteins were freed in 12 h fter mrna trnsfection, but the extr secreting of trget could lst for h. Considering tht humn PC cells showed unregulted production of DcR3 within 12 h, we found tht the time course of nti-dcr3 mab relese by DCs is idel for obstructing the DcR3 expressed by PC cells [8]. In the present study, we found tht DCs trnsfected with totl tumor RNA lone or together with nti-dcr3 mab mrna could induce tumor-specific cytotoxic T cells to recognize nd lyse tumor RNA-loded DCs nd tumor cells effectively. In comprison, no dmge in K562 cells were found, which implies tht the two mnners cn not only be PC-specific but lso eliminted the possibility of NK cell ctivity [2]. These dt lso indicte tht CTLs induced by DC-tumor- 826 Februry 7, 2017 Volume 23 Issue 5

11 Chen J et l. CTL responses to PC induced by DCs nti-dcr3 RNA were more powerful t indicting lysis thn the CTLs induced by DC-totl tumor RNA. These results demonstrte tht use of DCs co-trnsfected with RNA encoding humnized nti-dcr3 mab nd whole PC tumor-ntigens my be superior strtegy for designing DC-bsed tumor vccine. In ddition, it is known tht, like Fs-Fc ntibody, DcR3 cn block poptosis in Jurkt cells [21]. The more superior cytolytic function might be due to blockde of ctivtioninduced cell deth (AICD) in CTLs induced by DCtumor-nti-DcR3 RNA rther thn better CTL response genertion due to superior priming of CTL precursors by engineered DCs. This should be ddressed in future studies. An HLA llele tht mtches the trget cells nd tumor-specific CTLs is necessry. All PC ptients tht were included in this study were HLA-A2+, nd we re concerned with the function of HLA-A2 mong different HLA lleles. We found tht CTLs induced by DC-tumornti-DcR3 RNA nd DC-totl tumor RNA could deliver potent cytotoxicity towrds Cpn-2 cells. However, owing to HLA-A2 mismtching, AsPC-1 PC cells with HLA-A2- could not be lysed by HLAA2+ CTLs. This result indictes tht HLA-A2 my be key llele for presenting ntigens, nd PC-specific CTL immune response my be limited to MHC clss I ntigens. The induction of utoimmunity is one potentil problem tht my limit the ppliction of PC tumor nd nti-dcr3 mab RNA-trnsected DC vccines, prtly becuse RNAs loded with both norml ntigens nd tumor ntigens shre the sme ntigen presenttion pthwy when they re delivered to DCs [26,38], nd prtly becuse RNA-encoding nti-dcr3 mab in DCs my improve the risk of inducing utoimmunity just like systemic dministrtion of neutrlizing immunosuppressive fctor ntibody in TME [34]. Here, we dopted tissue cell enrichment by primry tumor cell culture, nd found tht CTLs stimulted by DC-totl tumor RNA lysed tumor cells (utologous primry cultured tumor cells nd PC cell lines) but not PBMCs (norml tissue cells). Menwhile, using mrna-trnsfected DCs to loclly deliver nti-dcr3 mab, no increse in non-specific bckground immune responses ginst control trget cells or the norml tissue PBMCs in vitro ws detected. On the bsis of these results, we nticipte tht locl delivery of nti- DcR3 mab by utilizing DCs trnsfected with RNA will bypss the dverse effects of utoimmune responses triggered by systemic delivery of mab. At the sme time, vccine-induced nti-tumor immune response incresed in ptients. These findings indicte tht hrmful utoimmunity with pthologicl results my not be n issue with this method. The mechnism by which DCs engineered to secrete nti-dcr3 mab ugments CTL response remins to be fully elucidted. In our study, we found tht the cell vibility of both DC-tumor-nti-DcR3 RNA nd DC-totl tumor RNA did not chnge significntly t the designted time points, with pproximtely 85% survivl when cultured lone (without DcR3 influence). On the contrry, when co-cultured with utologous tumor cells (with DcR3 influence), the vibility of DCtotl tumor RNA ws evidently lower thn tht of DCtumor-nti-DcR3 RNA. Furthermore, when co-cultured with tumor cells for 0-96 h, poptotic cells in DC-totl tumor RNA evidently incresed, wheres poptotic cells in DC-tumor-nti-DcR3 RNA were found to increse slowly nd mildly. Similr to the findings of You et l [39], results suggested tht enhncement of CTL responses induced by nti-dcr3 mab DCs my prtly be due to its powerful DcR3 blocking cpbility, which downregulted the poptosis of DCs induced by DcR3 nd incresed DC vibility t the site of T-cell ctivtion in the process of whole tumor-ntigen delivery. The whole totl tumor nd nti-dcr3 mab RNA electroportion likely cted s powerful tumor vccine which could effectively ctivte ntigen-specific T cells ginst PCs, just like Th1 cells [2]. This interprettion is supported by our observtions of high percentge of killing of tumor cells nd IFN-γ secretion of T cells. High expressions of cytokines, such s IL-12 nd IFN-γ, nd low expression of cytokine, such s IL-10, might be the reson why DC-tumor-nti-DcR3 RNA exhibited enhnced cpbility of inducing CTL responses. Th1 cells nd Th2 cells re two importnt T regultory (Treg) cells in the body. Trnsformtion of Treg cells from Th1 to Th2 is unique phenomenon in mlignnt tumors. Development of Th2 cells promotes long-term retention of cncer cells in the host body nd protects them from immune surveillnce nd ttck. Th1 cells nd Th2 cells cn both stimulte IFN-γ production, wheres Th2 cells preferentilly induced production of IL-4 compred to Th1 cells [39,40]. In this study, we clerly demonstrted tht CD4+ T cells induced by DCs co-trnsfected with totl tumor RNA nd nti- DcR3 mab mrna mrkedly decresed IL-4 secretion nd incresed IFN-γ production, indicting tht DCs engineered to secrete nti-dcr3 mab could increse the number of Th1 cells nd decrese Th2 cells. Ojim et l [41] nd Chen et l [2] reported tht DCs tht were loded with TAA could induce ntigen-specific CD4+Th1 cells nd such CD4+ Th1 cells plyed key role in the priming phse of CD8+ CTLs. In the present study, we showed tht both DC-tumor-nti-DcR3 RNA nd DC-totl tumor RNA cn ctivte not only tumorspecific CD4+ T cells but lso CD8+ T cells ssessed by IFN-γ relese. Besides, CD4+ T cells nd CD8+ T cells incubted with DC-tumor-nti-DcR3 RNA could produce more IFN-γ compred with those incubted with DC-totl tumor RNA. These results indicte tht djusting the Th1/Th2 cytokine network nd promoting the recovery of CD8+ nti-tumor cellulr immunity my be the other two mechnisms for engineering DCs to secrete nti-dcr3 mab to ugment CTL response. In summry, our results demonstrte tht DCs engineered to secrete nti-dcr3 ntibody cn ugment 827 Februry 7, 2017 Volume 23 Issue 5

12 Chen J et l. CTL responses to PC induced by DCs CTL responses ginst PC in vitro. The observed immune-enhncing effects my be prtly due to their cpbility of down-regulting DC poptosis nd djusting the Th1/Th2 cytokine network. Therefore, use of DCs engineered to secrete nti-dcr3 ntibody vccine my be n ttrctive nd promising therpeutic strtegy for ptient with PC. COMMENTS Bckground Pncretic cncer (PC) is considered s highly destructive humn mlignnt tumor without effective tretments. Dendritic cell (DC) tumor vccines hve emerged s n lterntive tretment mnner for dvnced PC. But tumor cells cn produce some immunosuppressive molecules to inhibit the function of tumor-ssocited cells, such s T lymphocytes nd DCs. Reserch frontiers DcR3, soluble protein secreted by tumor cells, is overexpressed in crcinom originting from the gstrointestinl trct system, including PC. DcR3 is regrded s n importnt immunosuppressive fctor in immune effector cells defect nd it is prticulrly importnt to neutrlize DcR3 protein secreted by tumor cells in cncer immunotherpy. Innovtions nd brekthroughs A new strtegy of delivering mab by trnsfecting DCs with the RNA encoding both nti-dcr3 mab nd the whole tumor-ntigens ws shown by this study. Furthermore, the DCs engineered to secrete nti-dcr3 mab could ugment cytotoxic T lymphocyte responses ginst PC in vitro nd the immuneenhncing effects my be prtly due to their cpbility of down-regulting poptosis of DCs nd djusting the Th1/Th2 cytokine network. Applictions This study lys good foundtion for further investigtion of tumor DC vccine trgeting DcR3 protein ginst PC. Terminology As tumor necrosis fctor receptor superfmily s member, DcR3 works s decoy receptor for Fs lignd, LIGHT nd TNF-like molecule 1A to neutrlize their cytotoxic nd regultory functions. Peer-review This mnuscript hs shown tht DCs engineered to secrete nti-dcr3 ntibody cn stimulte cytotoxic T lymphocyte responses ginst pncretic cncer cells in vitro. It provided importnt contribution to development of immunotherpy for pncretic cncers. The proposed method is convincing. REFERENCES 1 Siegel RL, Miller KD, Jeml A. Cncer sttistics, CA Cncer J Clin 2015; 65: 5-29 [PMID: DOI: / cc.21254] 2 Chen J, Guo XZ, Li HY, Wng D, Sho XD. Comprison of cytotoxic T lymphocyte responses ginst pncretic cncer induced by dendritic cells trnsfected with totl tumor RNA nd fusion hybrided with tumor cell. Exp Biol Med (Mywood) 2015; 240: [PMID: DOI: / ] 3 Hidlgo M. Pncretic cncer. N Engl J Med 2010; 362: [PMID: DOI: /NEJMr ] 4 Li D, O Reilly EM. Adjuvnt nd Neodjuvnt Therpy for Pncretic Cncer. Surg Oncol Clin N Am 2016; 25: [PMID: DOI: /j.soc ] 5 Arsln C, Ylcin S. Current nd future systemic tretment options in metsttic pncretic cncer. 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13 Chen J et l. CTL responses to PC induced by DCs 21 Wng W, Zhng M, Sun W, Yng S, Su Y, Zhng H, Liu C, Li X, Lin L, Kim S, Okunieff P, Zhng Z, Zhng L. Reduction of decoy receptor 3 enhnces TRAIL-medited poptosis in pncretic cncer. PLoS One 2013; 8: e74272 [PMID: DOI: /journl.pone ] 22 Edge SB, Compton CC. The Americn Joint Committee on Cncer: the 7th edition of the AJCC cncer stging mnul nd the future of TNM. Ann Surg Oncol 2010; 17: [PMID: DOI: /s ] 23 Wng K, Zhou Q, Guo AL, Xu CR, An SJ, Wu YL. An utologous therpeutic dendritic cell vccine trnsfected with totl lung crcinom RNA stimultes cytotoxic T lymphocyte responses ginst non-smll cell lung cncer. Immunol Invest 2009; 38: [PMID: DOI: / ] 24 Wu CF, M ZS, Wng XL, Yng JL, Yng Y, Zhng Y, Tumen WL, Qio JH. Preprtion nd identifiction of monoclonl ntibodies ginst humn DcR3. Xibo Yu Fenzi Minyixue Zzhi 2008; 24: [PMID: ] 25 Zhu M, Xu W, Su H, Hung Q, Wng B. Addition of CpG ODN nd Poly (I: C) to stndrd mturtion cocktil genertes monocytederived dendritic cells nd induces potent Th1 polriztion with migrtory cpcity. Hum Vccin Immunother 2015; 11: [PMID: DOI: / ] 26 Chen J, Guo XZ, Li HY, Liu X, Ren LN, Wng D, Zho JJ. Genertion of CTL responses ginst pncretic cncer in vitro using dendritic cells co-trnsfected with MUC4 nd survivin RNA. Vccine 2013; 31: [PMID: DOI: / j.vccine ] 27 Chen J, Li HY, Wng D, Zho JJ, Guo XZ. Humn dendritic cells trnsfected with mplified MUC1 mrna stimulte cytotoxic T lymphocyte responses ginst pncretic cncer in vitro. J Gstroenterol Heptol 2011; 26: [PMID: DOI: /j x] 28 Lin XY, He CD, Xio T, Jin X, Chen J, Wng YK, Liu M, Wng KB, Jing Y, Wei HC, Chen HD. Acitretin induces poptosis through CD95 signlling pthwy in humn cutneous squmous cell crcinom cell line SCL-1. J Cell Mol Med 2009; 13: [PMID: DOI: /j x] 29 Miyzw M, Iwhshi M, Ojim T, Ktsud M, Nkmur M, Nkmori M, Ued K, Nk T, Hyt K, Iid T, Ymue H. Dendritic cells denovirlly-trnsduced with full-length mesothelin cdna elicit mesothelin-specific cytotoxicity ginst pncretic cncer cell lines in vitro. Cncer Lett 2011; 305: [PMID: DOI: /j.cnlet ] 30 Constntino J, Gomes C, Flcão A, Cruz MT, Neves BM. Antitumor dendritic cell-bsed vccines: lessons from 20 yers of clinicl trils nd future perspectives. Trnsl Res 2016; 168: [PMID: DOI: /j.trsl ] 31 Bi C, Connolly B, Metzker ML, Hillird CA, Liu X, Sndig V, Sodermn A, Gllowy SM, Liu Q, Austin CP, Cskey CT. Overexpression of M68/DcR3 in humn gstrointestinl trct tumors independent of gene mplifiction nd its loction in fourgene cluster. Proc Ntl Acd Sci USA 2000; 97: [PMID: DOI: /pns ] 32 Hung MT, Chen ST, Wu HY, Chen YJ, Chou TY, Hsieh SL. DcR3 suppresses influenz virus-induced mcrophge ctivtion nd ttenutes pulmonry inflmmtion nd lethlity. J Mol Med (Berl) 2015; 93: [PMID: DOI: / s ] 33 Shi G, Wu Y, Zhng J, Wu J. Deth decoy receptor TR6/DcR3 inhibits T cell chemotxis in vitro nd in vivo. J Immunol 2003; 171: [PMID: DOI: / jimmunol ] 34 Glluzzi L, Vcchelli E, Fridmn WH, Glon J, Sutès-Fridmn C, Trtour E, Zucmn-Rossi J, Zitvogel L, Kroemer G. Tril Wtch: Monoclonl ntibodies in cncer therpy. Oncoimmunology 2012; 1: [PMID: DOI: /onci ] 35 Sunthrlingm G, Perry MR, Wrd S, Brett SJ, Cstello-Cortes A, Brunner MD, Pnoskltsis N. Cytokine storm in phse 1 tril of the nti-cd28 monoclonl ntibody TGN1412. N Engl J Med 2006; 355: [PMID: DOI: / NEJMo063842] 36 Postel-Viny S, Aspeslgh S, Lnoy E, Robert C, Sori JC, Mrbelle A. Chllenges of phse 1 clinicl trils evluting immune checkpoint-trgeted ntibodies. Ann Oncol 2016; 27: [PMID: DOI: /nnonc/mdv550] 37 Weinstock M, McDermott D. Trgeting PD-1/PD-L1 in the tretment of metsttic renl cell crcinom. Ther Adv Urol 2015; 7: [PMID: DOI: / ] 38 Gnji A, Vrsteh A, Snkin M. Aptmers: new rrows to trget dendritic cells. J Drug Trget 2016; 24: 1-12 [PMID: DOI: / X ] 39 You RI, Chng YC, Chen PM, Wng WS, Hsu TL, Yng CY, Lee CT, Hsieh SL. Apoptosis of dendritic cells induced by decoy receptor 3 (DcR3). Blood 2008; 111: [PMID: DOI: /blood ] 40 Hillyer P, Rviv N, Gold DM, Dougherty D, Liu J, Johnson TR, Grhm BS, Rbin RL. Subtypes of type I IFN differentilly enhnce cytokine expression by suboptimlly stimulted CD4(+) T cells. Eur J Immunol 2013; 43: [PMID: DOI: /eji ] 41 Ojim T, Iwhshi M, Nkmur M, Mtsud K, Nkmori M, Ued K, Nk T, Ishid K, Primus FJ, Ymue H. Successful cncer vccine therpy for crcinoembryonic ntigen (CEA)- expressing colon cncer using geneticlly modified dendritic cells tht express CEA nd T helper-type 1 cytokines in CEA trnsgenic mice. Int J Cncer 2007; 120: [PMID: DOI: /ijc.22298] P- Reviewer: Chhbr A, Ferrnte A, MtsudY, Trnwski AS S- Editor: Qi Y L- Editor: Filipodi E- Editor: Wng CH 829 Februry 7, 2017 Volume 23 Issue 5

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