Synthetic biology has advanced the design of standardized transcription controldevices that programme cellular behaviour. By coupling synthetic signalling cascade-and transcription factor-based gene switches with reverse and differentialsensitivity to the licensed food additive vanillic acid, we designed a syntheticlineage-control network combining vanillic acid-triggered mutually exclusiveexpression switches for the transcription factors Ngn3 and Pdx1  with the concomitantinduction of MafA . This designer network consisting of different network topologiesorchestrating the timely control of transgenic and genomic Ngn3, Pdx1 and MafAvariants is able to programme human induced pluripotent stem cells (hIPSCs)-derivedpancreatic progenitor cells into glucose-sensitive insulin-secreting beta-likecells, whose glucose-stimulated insulin-release dynamics are comparable to humanpancreatic islets. Synthetic lineage-control networks may provide the missing linkto genetically programme somatic cells into autologous cell phenotypes forregenerative medicine. Synthetic biology offers the potential for the design andimplementation of rationally designed, complex genetic programmes. Here the authorsdesign a genetic network to trigger the differentiation of patient derived IPSCs intobeta-like cells. Pdx1 are associated with pancreasagenesis in mouse and humans5111Cell-fate decisions during development are regulated by various mechanisms, includingmorphogen gradients, regulated activation and silencing of key transcription factors,microRNAs, epigenetic modification and lateral inhibition. The latter implies that thedecision of one cell to adopt a specific phenotype is associated with the inhibition ofneighbouring cells to enter the same developmental path. In mammals, insights into therole of key transcription factors that control development of highly specialized organslike the pancreas were derived from experiments in mice, especially various geneticallymodified animals123invitro generation of bonafide insulin-secreting beta-like cells that aresuitable for transplantation have been the result of sophisticated trial-and-errorstudies elaborating timely addition of complex growth factor and small-molecule compoundcocktails to human pancreatic progenitor cells32333536In recent years, synthetic biology has significantly advanced the rational design ofsynthetic gene networks that can interface with host metabolism, correct physiologicaldisturbances18192125272929The differentiation pathway from pancreatic progenitor cells to glucose-sensitiveinsulin-secreting pancreatic beta-cells combines the transient mutuallyexclusive expression switches of Ngn3 (OFF-ON-OFF) and Pdx1 (ON-OFF-ON) with theconcomitant induction of MafA (OFF-ON) expression10Sα) and adenylyl cyclase to produce acyclic AMP (cAMP) second messenger surgeCRE) containing CREB1-specific cAMP response elements and pCK53(PCRE-SEAP-pASV40) into human mesenchymal stem cells(hMSC-TERT) confirmed the vanillic acid-adjustable secreted alkaline phosphatase(SEAP) induction of the designer cascade , which bindsand activates vanillic acid-responsive promoters  at low and medium vanillic acid levels(<2 μM). At high vanillic acid concentrations(>2 μM), VanA1 dissociates from P1VanO2,which results in the dose-dependent repression of transgene expressionpMG250 (PSV40-VanA1-pASV40) andpMG252 (P1VanO2-SEAP-pASV40) into hMSC-TERTcorroborated the fine-tuning of the vanillic acid-repressible SEAP expression(The core of the synthetic lineage-control network consists of two transgenecontrol devices that are sensitive to the food component and licensed foodadditive vanillic acid. These devices are a synthetic vanillic acid-inducible(ON-type) signalling cascade that is gradually induced by increasing thevanillic acid concentration and a vanillic acid-repressible (OFF-type) geneswitch that is repressed in a vanillic acid dose-dependent manner . The desntsCRE; . The co-pression.pCI-MOR9-1;PhCMV-MOR9-1-pASV40; pSP1,PCRE-VanA1-pASV40) and the gene switch in the same cell,the network executes a band-pass filter SEAP expression profile when exposed toincreasing concentrations of vanillic acid for endocrine specification and pSP17(PCREm-Pdx1cm-2A-MafAcm-pASV40)for maturation of developing beta-cells  that drives expression of a codon-modifiedNgn3cm, the nucleic acid sequence of which isdistinct from its genomic counterpart (Ngn3g) to allowfor quantitative reverse transcription–PCR (qRT–PCR)-baseddiscrimination. In the opposite direction, P3VanO2 transcribesmiR30Pdx1g-shRNA, which exclusively targets genomic Pdx1(Pdx1g) transcripts for RNA interference-baseddestruction and is linked to the production of a blue-to-red medium fluorescenttimerin situ.pSP17 contains a dicistronic expression unit in which the modifiedhigh-tightness and lower-sensitivity PCREm promoter (see below)drives co-cistronic expression of Pdx1cm andMafAcm, which are codon-modified versions producingnative transcription factors that specifically differ from their genomiccounterparts  intheir nucleic acid sequence. After individual validation of the vanillicacid-controlled expression and functionality of all network components , pSP1(PCRE-VanA1-pASV40), pSP12(pASV40-Ngn3cm←P3VanO2→mFT-miR30Pdx1g-shRNA-pASV40)and pSP17(PCREm-Pdx1cm-2A-MafAcm-pASV40)into hIPSC-derived pancreatic progenitor cells, the synthetic lineage-controlnetwork should override random endogenous differentiation activities and executethe pancreatic beta-cell-specific differentiation programme in a vanillic acidremote-controlled manner. To confirm that the lineage-control network operatesas programmed, we cultivated network-containing and pEGFP-N1-transfected(negative-control) cells for 4 days at medium (2 μM) and then 7days at high (400 μM) vanillic acid concentrations and profiledthe differential expression dynamics of all of the network components and theirgenomic counterparts as well as the interrelated transcription factors andhormones in both whole populations and individual cells at days 0, 4, 11 and 14 and the notch-signallingcomponents Hes1 (hairy and enhancer of split-1) and Dll1(delta-like 1), which manage lateral inhibition  expression levels innetwork-transfected cells remained identical to randomly differentiating cells: medium vanillic acidconcentrations trigger the MOR9-1-based designer cascade and inducePpression . Ngn3cm  OFF-ON;  as welldays 4; .After sCRE-drivenVanA1 expression at medium vanillic acid concentrations,VanA1 co-induces the P3VanO2-driven expression ofNgn3cm and miR30Pdx1g-shRNA. As Ngn3cm andmiR30Pdx1g-shRNA levels increase, miR30Pdx1g-shRNAprogrammes the exclusive destruction of genomic Pdx1(Pdx1g) transcripts, which results in areduction of Pdx1g levels , which then leads tofeed-forward loop-type signal amplification and increased expression kinetics ofmiR30Pdx1g-shRNA compared with Pdx1cm  expression plug-in . During the co-induction ofNgn3cm and miR30Pdx1g-shRNA as well asmiR30Pdx1g-shRNA-mediated Pdx1gknockdown at medium vanillic acid concentrations, the cells first fluoresce inblue and then simultaneously in blue and red as the mFT matures :Following the MOR9-1-mediated activation of P ON-OFF; .Becausew day 4; . At high1g-shRNA . The trah Pdx1cm . The tim matures . After se in red. Aftertn yellow.CREm has been optimized fortightness and decreased vanillic acid sensitivitycm is repressed at medium vanillic acid concentrations, as isrequired for the early phase of the differentiation processcm is induced and Pdx1g is repressed : Because Pd day 4;. At high OFF-ON; .MafAcm  day 11; .To validate the beta-like cells genetically programmed from hIPSC-derivedpancreatic progenitor cells using the synthetic lineage-control network, wecompared their phenotype to cells produced using thegrowth-factor/chemical-based differentiation technique  as well 3233344748MafA, NeuroD, Pax4 and Pdx1,whereas NeuroD, Pax4 and Pdx1 were expressed at lowerlevels in beta-like cells produced by the growth-factor/chemical-baseddifferentiation technique (Gck (glucokinase) and G6pc2(glucose-6-phosphatase 2) as well as the insulin-processing factor Pcsk1(prohormone convertase 1) were expressed at higher levels in beta-like cellsproduced using the lineage-control network compared with those resulting fromthe growth-factor/chemical-based differentiation technique , Irx2 (Iroquois homeobox2) and Acox2 (Acyl-CoA oxidase 2) were expressed at lower levels, whereasmarkers specific for mature beta-cells such as FoxA1 (Forkhead box A1(ref. Gcgr (Glucagon receptor) wereexpressed at a higher levels in lineage- than growth-factor/chemical-controlledbeta-like cells (1 as well as insulin (C-peptide), whereas onlyvery few VanA1-containing cells co-stained for glucagon andsomatostatin  thetranscription factors echnique . (ii) Liechnique . (iii) Technique . (iv) Inc islets . Theexpke cells. (v) Mar A1ref.  and Gcgrke cells . (vi) Fltostatin . These dtostatin . By conttostatin . (vii) Itostatin . (viii) tostatin . In additostatin .Overall, all of these characteristics corroborate our finding that the syntheticlineage-control network is able to programme the cell fate of hIPSC-derivedpancreatic progenitor cells and specifically differentiate them intoglucose-sensitive insulin-secreting pancreatic beta-like cells. Indeed, electronmicrographs of these lineage-controlled beta-like cells show the typicalinsulin-storage vesicles that are found in mature pancreatic beta cells .Multicellular organisms, including humans, consist of a highly structured assembly ofa multitude of specialized cell phenotypes that originate from the same zygote andhave traversed a preprogrammed multifactorial developmental plan that orchestratessequential differentiation steps with high precision in space and time19In vitro differentiation of hESC-derived pancreatic progenitor cells intobeta-like cells is more challenging and has been achieved recently by a complexmedia formulation with chemicals and growth factors3233Despite significant progress in regenerative medicine and the availability of stemcells, the design of protocols that replicate natural differentiation programmes andprovide fully functional cell mimetics remains challenging293154hIPSCs have become a promising alternative to hESCs; however, their use remainsrestricted in many countries3757Recent studies applying similar hESC-based differentiation protocols to hIPSCs haveproduced cells that release insulin in response to high glucose32333235in situ production of therequired transcription factors, (ii) enable simultaneous control of ectopic andchromosomally encoded transcription factor variants, (iii) tap into endogenouspathways and not be limited to cell-surface input, (iv) display improvedreversibility that is not dependent on the removal of exogenous growth factors viaculture media replacement, (v) provide lateral inhibition, thereby reducing therandom differentiation of neighbouring cells and (vi) enable trigger-programmableand (vii) precise differential transcription factor expression switches.Rather than exposing hIPSCs to a refined compound cocktail that triggers the desireddifferentiation in a fraction of the stem cell population, we chose to design asynthetic lineage-control network to enable single input-programmabledifferentiation of hIPSC-derived pancreatic progenitor cells into glucose-sensitiveinsulin-secreting beta-like cells. In contrast with the use ofgrowth-factor/chemical-based cocktails, synthetic lineage-control networks areexpected to (i) be more economical because of The synthetic lineage-control network that precisely replicates the endogenousrelative expression dynamics of the transcription factors Pdx-1, Ngn3 and MafArequired the design of a new network topology that interconnects a syntheticsignalling cascade and a gene switch with differential and opposing sensitivity tothe food additive vanillic acid. This differentiation device provides differentband-pass filter, time-delay and feed-forward amplifier topologies that interfacewith endogenous positive-feedback loops to orchestrate the timely expression andrepression of heterologous and chromosomally encoded Ngn3, Pdx1 and MafA variants.The temporary nature of the engineering intervention, which consists of transienttransfection of the genetic lineage-control components in the absence of anyselection, is expected to avoid stable modification of host chromosomes andalleviate potential safety concerns. In addition, the resulting beta-cell mass couldbe encapsulated inside vascularized microcontainers14151661The hIPSC-derived beta-like cells resulting from this trigger-induced syntheticlineage-control network exhibited glucose-stimulated insulin-release dynamics andcapacity matching the human physiological range and transcriptional profiling, flowcytometric analysis and electron microscopy corroborated the lineage-controlled stemcells reached a mature beta-cell phenotype. In principle, the combination of hIPSCsderived from the adipose tissue of a 50-year-old donor3066Capitalizing on the design principles of synthetic biology, we have successfullyconstructed and validated a synthetic lineage-control network that replicates thedifferential expression dynamics of critical transcription factors and mimicks thenative differentiation pathway to programme hIPSC-derived pancreatic progenitorcells into glucose-sensitive insulin-secreting beta-like cells that compare withhuman pancreatic islets at a high level. The design of input-triggered syntheticlineage-control networks that execute a preprogrammed sequential differentiationagenda coordinating the timely induction and repression of multiple genes couldprovide a new impetus for the advancement of developmental biology and regenerativemedicine.pCI-MOR9-1 encodesconstitutively expressed vanillic acid receptor ; pSP1 encodescAMP-responsive expression cassette for the vanillic acid-dependenttransactivator ; pSP12 contains avanillic acid-responsive Ngn3cm, mFT and miR30Pdx1g-shRNAexpression unit(pASV40←Ngn3cm-P3VanO2→mFT-miR30Pdx1g-shRNA-pASV40)and pSP17 contains a cAMP-responsive dicistronic Pdx1cm andMafAcm expression unit(PCREm-Pdx1cm-2A-MafAcm-pASV40).Comprehensive design and construction details for all expression vectors areprovided in 2. The hIPSCs were derived from the adipose tissue of a50-year-old donor−1 dispase (STEMCELL Technologies).For the transfection of hMSC-TERT, 2 × 105 cells wereseeded per well on a six-well plate 12 h before transfection andincubated for 24 h with 400 μl of a DNA-polyethyleneimine(PEI) mixture that was produced by incubating 6 μl PEI  with2 μg of total DNA, vortexing for 5 s and incubating for15 min at 22 °C. Before transfection, the hIPSCs-derivedpancreatic progenitor cells were dissociated using 0.5 ml of StemProAccutase Cell Dissociation Reagent (Invitrogen) and reseeded into 12-wellplates. For the transfection, 3 μg of total DNA was transfectedinto cells cultivated in each well of a 12-well plate using Lipofectamine LTXReagent with PLUS Reagent (Invitrogen). The plasmids  were co-transfected at a ratio of1:1:1:1 for hIPSCs (1:0.01:1:1 for hMSC-TERT). The transfection efficiency wasdetermined via FACS analysis using pEGFP-N1-transfected, randomlydifferentiating cells as a control (−1 exendin-4 (TocrisBioscience), 10 mM nicotinamide (Sigma-Aldrich) and 40 μMβ-mercaptoethanol. Subsequently, the culture medium was replaced by thesame supplemented RPMI devoid of Y-27632 and the cells were transfected with3 μg pSP26(PhINS-DsRed-Express-pASV40) using Lipofectamine LTXwith Plus Reagent (Invitrogen). For FACS-mediated single-cell sorting of thelineage- and growth-factor/chemical-controlled beta-like cells (day 11),hIPSC-derived pancreatic progenitor cells (day 0) were co-transfected with3 μg of total DNA . The parental vector pcDNA3.1(+) was used as afiller plasmid when replacing functional components for control purposes. Forthe dose–response experiments, the transfected cells were trypsinized andtransferred into the wells of a 96-well plate (104 cells perwell) containing 100 μl DMEM supplemented with differentconcentrations of vanillic acid. SEAP expression was profiled after 48 h.To compare vanillic acid-triggered lineage control with independent expressioncontrol of the pancreatic transcription factors Ngn3, Pdx1, MafA by differentantibiotic-responsive gene switches, we co-transfected hMSC-TERT withpCI-MOR9-1, pSP1, pSP12 and pSP17 for thelineage-control network, with pWW35, pSP36, pMF156 andpSP39 for macrolide-responsive Pdx1 and MafA (EOFF) aswell as streptogramin-responsive Ngn3 expression (PIPOFF) or withpWW35, pSP37, pMF156, pSP39, pTet-ON andpSP4 for macrolide-responsive Pdx1 (EOFF),streptogramin-responsive Ngn3 (PIPOFF) and tetracycline-responsiveMafA (TETON) expression, and incubated the cells in the presence ofvanillic acid , erythromycin ,doxycycline  or pristinamycin .Human mesenchymal stem cells transgenic for the catalytic subunit of humantelomerase hMSC-TERT were cul control . Randoml−1) and Wnt3A(25 ng ml−1). Then, the cells werecultivated for another 48 h in RPMI containing Activin A(100 ng ml−1), bFGF(5 ng ml−1), BMP-4(0.25 ng ml−1) and VEGF165(10 ng ml−1). Subsequently, the cellswere cultivated for another 48 h in serum-free differentiation (SFD)medium , 1-thioglycerol(0.45 μM)) containing Activin A(100 ng ml−1), bFGF(5 ng ml−1), BMP-4(0.25 ng ml−1) and VEGF165(10 ng ml−1). Step 2 (primitive gut tubecells): The endoderm cells resulting from step 1 were cultivated for 72 hin SFD medium containing Wnt3A (3 ng ml−1),Noggin (50 ng ml−1) and FGF-10(50 ng ml−1). Step 3 (pancreaticprogenitor cells): The primitive gut tube cells resulting from step 2 werecultivated for 72 h in DMEM containing 1% (vol/vol) vitamin A-freeB−27 serum-free supplement, ascorbic acid(50 μg ml−1), KAAD-cyclopamine(0.25 μM), retinoic acid (2 μM) and FGF-10(50 ng ml−1). Step 4 (pancreaticprogenitor cells): The pancreatic progenitor cells resulting from step 3 werecultivated for 72 h in DMEM containing 1% (vol/vol) vitamin A-freeB−27 serum-free supplement, ascorbic acid(50 μg ml−1), retinoic acid(100 nM), FGF-7 (50 ng ml−1) andepidermal growth factor .Step 5 (endocrine progenitor cells): The pancreatic progenitor cells resultingfrom step 4 were treated for 4 h with Y-27632 (10 μM),dissociated for 5 min using 0.5 ml of StemPro Accutase andincubated for 72 h in suspension plates to facilitate the formation ofaggregates in DMEM containing 1% (vol/vol) vitamin A-free B−27serum-free supplement, ascorbic acid(50 μg ml−1), retinoic acid(50 nM), thyroid hormone T3 (1 μM), Alk5 inhibitor(10 μM), Noggin (50 ng ml−1),KAAD-cyclopamine (0.25 μM) and gamma secretase inhibitor(1 μM). Step 6 (maturing beta-like cells): The endocrineprogenitor cells resulting from step 5 were cultivated for 72 h in DMEMcontaining 1% (vol/vol) vitamin A-free B−27 serum-free supplement,ascorbic acid (50 μg ml−1), retinoicacid (25 nM), thyroid hormone T3 (1 μM), Noggin(50 ng ml−1), Alk5 inhibitor(10 μM) and gamma secretase inhibitor (1 μM). Step 7(beta-like cells): The maturing beta-like cells resulting from step 6 werecultivated for 5 days in low-glucose DMEM (5 mM glucose) supplementedwith 10% (vol/vol) FCS and containing 1 × non-essential amino acidsolution, thyroid hormone T3 (1 μM) and Alk5 inhibitor(10 μM). For the lineage-control network, 12-well suspensionplates (Greiner Bio-one GmbH.) were used from day 0 onwards and hIPSC-derivedendodermal cells were cultivated in noggin- and gamma-secretase inhibitor-freeculture medium (to prevent premature endocrine differentiation beforetransfection of the lineage-control network) supplemented with epidermal growthfactor (to trigger expression of Nkx6.1 in pancreatic progenitor cells). Afterdissociation and transfection of the pancreatic progenitor cells with thelineage-control network, the engineered cells spontaneously form aggregates andfollowing addition of thyroid hormone T3 (triiodothyronine) and retinoic acidthey differentiate into endocrine progenitor cells and mature into beta-likecells following addition of Alk5 inhibitor , vanillic acid, ascorbic acid, bovine serum albumin,1-thioglycerol, thyroid hormone T3 and retinoic acid were purchased fromSigma-Aldrich. The gamma secretase inhibitor L-685,458 was obtained from TocrisBioscience, Rock inhibitor Y-27632 was obtained from STEMCELL Technologies andAlk5 inhibitor was obtained from Enzo Lifesciences.The hIPSCs were differentiated to pancreatic progenitor cells and beta-like cellsusing the growth-factor/chemical-based differentiation techniques describedbelow32333455nhibitor . RandomlAt different time points of the differentiation . The activity of Photinus pyralis firefly luciferase (FLuc)was quantified using a Luciferase Assay System (Promega).The production of human placental SEAP was quantified in cell culturesupernatants using a Ct) wasdetermined and normalized to the endogenous glyceraldehyde 3-phosphatedehydrogenase transcript. The fold change for each transcript relative to thecontrol was calculated using the comparative Ct methodTotal RNA of lineage-controlled hIPSC population, FACS-sorted hIPSC-derivedbeta-like cells, engineered hMSC-TERT and human islets was isolated using the ZRRNA MiniPrep kit (Zymo Research) and TURBO DNase (Invitrogen). qRT–PCRwith total RNA was performed using SuperScript II Reverse Transcriptase(Invitrogen) and TaqMan Fast Advanced Mastermix (Invitrogen) with thecorresponding TaqMan Gene Expression Assays  or SYBR pSP26(PhINS-DsRed-Express-pASV40) and the resultingbeta-like cells were sorted (day 14) using a Becton Dickinson LSRII Fortessaflow cytometer (Becton Dickinson) equipped for DsRed  detection. ForFACS-mediated analysis and single-cell sorting of hIPSC-derived beta-like cellsdifferentiated using the growth-factor/chemical-based differentiation technique(day 11), the respective treated population (day 0) was co-transfected with3 μg of total DNA  for the lineage-controlnetwork;  for the growth-factor/chemical-baseddifferentiation technique and the resulting beta-like cells were sorted usingDsRed  detection and EGFP  detection while excluding dead cells and cell doublets.Total RNA extracted from the sorted beta-like cells was used for qRT–PCR.For FACS analysis, we used the same antibodies and dilutions as forimmunostaining. In brief, cells were detached (1 × 106cells per ml) using 0.5 ml StemPro Accutase Cell Dissociation Reagent(Invitrogen) and fixed with 4% (wt/vol) paraformaldehyde in PBS overnightat 4 °C, permeabilized for 10 min with 0.1% (vol/vol)Triton X-100, washed once in 2 ml PBS and sequentially labelled for1 h at 37 °C with specific primary and secondary antibodies,with a PBS washing step in between. The cell populations were analysed using aBecton Dickinson LSRII Fortessa flow cytometer (Becton Dickinson) equipped forEGFP , DsRed  or allophycocyanin  detection and set to exclude dead cells and cell doublets. Unstainedcell populations were used as negative controls.For FACS-mediated analysis and single-cell sorting of randomly differentiatingand lineage-controlled hIPSC-derived beta-like cells (day 11), the cellpopulations was co-transfected with Human islets and beta-like cells differentiated using the lineage-control network(day 11) were dissociated into single cells using 0.5 ml of StemProAccutase Cell Dissociation Reagent (Invitrogen) and prepared forelectron-microscopic analysis using a standard procedure3,4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mMMgSO4, 2.5 mM CaCl2, 10 mM HEPES, pH7.4; Sigma-Aldrich) and incubated for 30 min in low-glucose Krebs-Ringer Bicarbonate Buffer supplemented with100 μM 3-isobutyl-1-methylxanthine (Sigma-Aldrich). The culturewas then switched to intermediate glucose (10 mM) for 30 min andthen high-glucose (20 mM) Krebs-Ringer Bicarbonate Buffer for another30 min. Thereafter, the culture was switched to high-glucose(20 mM) and KCl (30 mM)-supplemented Krebs-Ringer BicarbonateBuffer for another 30 min. The intracellular  and secreted isoforms of the connecting peptide (C-peptide) producedduring proinsulin processing were quantified using the ultrasensitive humanC-peptide ELISA . The values were normalized to thetotal intracellular protein content and the number of glucose-sensitiveinsulin-secreting cells using a Bio-Rad protein assay (Bio-Rad Laboratories).For the extended culture, beta-like cells (day 11) were continuously cultivatedfor 4 weeks in DMEM (Invitrogen) containing 10% (vol/vol) FCS,5 mM glucose, 400 μM vanillic acid (Sigma-Aldrich),1 μM thyroid hormone T3 (Sigma-Aldrich) and 10 μMAlk5 inhibitor before glucose-stimulated insulin release was quantified.Human islets and beta-like cells either differentiated by the syntheticlineage-control network or using the growth-factor/chemical-baseddifferentiation technique (day 11) were washed in 0.25 ml Krebs-RingerBicarbonate Buffer  using GraphPad Prism6 software (GraphPad Software Inc.).All data represent the means±s.d. of three independent experiments withtwo or three samples per experiment. Group comparisons were analysed by theStudent's How to cite this article: Saxena, P. et al. A programmable syntheticlineage-control network that differentiates human IPSCs into glucose-sensitiveinsulin-secreting beta-like cells. Nat. Commun. 7:11247 doi:10.1038/ncomms11247 (2016).Supplementary Figures 1-20, Supplementary Tables 1-3 and SupplementaryReferencesTime-lapse fluorescence microscopy of hMSC-TERT cotransfected with thelineagecontrol network vectors pCI-MOR9-1 (PhCMV-MOR9-1-pA), pSP1(PCRE-VanA1-pA), pSP12(pA-Ngn3cm←P3VanO2→mFTmiR30Pdx1g-shRNA-pA) and pSP24 and grown for 59h in the presenceof medium (2μM) vanillic acid concentration.Time-lapse fluorescence microscopy of hMSC-TERT cotransfected with thelineagecontrol network vectors pCI-MOR9-1 (PhCMV-MOR9-1-pA), pSP1(PCRE-VanA1-pA), pSP12(pA-Ngn3cm←P3VanO2→mFTmiR30Pdx1g-shRNA-pA) and pSP24 and grown for 48h in the presenceof high (400μM) vanillic acid concentration.


In both ischemic (ICM) and non-ischemic (NICM) cardiomyopathy late gadolinium enhancement (LGE) is an important cardiovascular magnetic resonance (CMR) technique. LGE CMR, traditionally performed in 2D during multiple breath-holds (MB), is challenging for vulnerable patients and subject to slice misregistration. Therefore, LGE CMR during free-breathing (FB) is more robust and enables to perform 3D acquisition, increase resolution and apply phase-sensitive inversion recovery (PSIR). We developed and clinically tested a high spatial resolution PSIR LGE sequence using a respiratory navigator in both ICM and NICM patients. Reliability and image quality were compared to a multiple breath-hold 3D approach (MB-3D).Approval was waived by the institutional ethical committee. Between May 2012 and November 2013 FB-3D and MB-3D sequences were acquired in patients suspected of ICM and NICM. Data of 48 patients  were retrospectively collected. LGE sequences were acquired using a 3.0 Tesla MR system. For both FB-3D (1.68 x 1.68 x 3.4 mm) and MB-3D (1.86 x 2.8 x 10 mm) a PSIR sequence was used. Multiplanar reformats with high resolution  and low resolution  were constructed. LGE mass and image quality  were compared using the Friedman test, Wilcoxon signed rank test, spearman correlation and Bland-Altman analysis.34 ICM patients  and 14 NICM were included . In figures Free-breathing 3D phase-sensitive inversion recovery LGE CMR enables reliable myocardial scar tissue assessment with significantly improved image quality as compared with multiple breath-hold 3D imaging in ischemic and non-ischemic cardiomyopathy.No funding.


The recently identified prostaglandin E receptor (EP) 4–associated protein (EPRAP) is essential for an anti-inflammatory function of EP4 signaling in macrophages in vitro. To investigate the in vivo roles of EPRAP, we examined the effects of EPRAP on colitis and colitis-associated tumorigenesis. In mice, EPRAP deficiency exacerbated colitis induced by dextran sodium sulfate (DSS) treatment. Wild-type (WT) or EPRAP-deficient recipients transplanted with EPRAP-deficient bone marrow developed more severe DSS-induced colitis than WT or EPRAP-deficient recipients of WT bone marrow. In the context of colitis-associated tumorigenesis, both systemic EPRAP null mutation and EPRAP-deficiency in the bone marrow enhanced intestinal polyp formation induced by azoxymethane (AOM)/DSS treatment. Administration of an EP4-selective agonist, ONO-AE1-329, ameliorated DSS-induced colitis in WT, but not in EPRAP-deficient mice. EPRAP deficiency increased the levels of the phosphorylated forms of p105, MEK, and ERK, resulting in activation of stromal macrophages in DSS-induced colitis. Macrophages of DSS-treated EPRAP-deficient mice exhibited a marked increase in the expression of pro-inflammatory genes, relative to WT mice. By contrast, forced expression of EPRAP in macrophages ameliorated DSS-induced colitis and AOM/DSS-induced intestinal polyp formation. These data suggest that EPRAP in macrophages functions crucially in suppressing colonic inflammation. Consistently, EPRAP-positive macrophages were also accumulated in the colonic stroma of ulcerative colitis patients. Thus, EPRAP may be a potential therapeutic target for inflammatory bowel disease and associated intestinal tumorigenesis.Prostaglandin E in vivo. In addition, EPRAP was localized in stromal macrophages of ulcerative colitis patients. This study revealed that EPRAP in macrophage participates critically in EP4 receptor signaling-mediated inhibition of intestinal inflammation. The macrophage EP4–EPRAP axis thus comprises a novel therapeutic target for IBD.Inflammatory bowel disease (IBD) is one of the most prevalent and serious gastrointestinal diseases in Western countries and associates with cancer development. EP4 receptor signaling can suppress intestinal inflammation and shows promise as a target for the development of novel therapies for IBD. To date, however, the lack of detailed molecular targets has hampered the development of effective drugs. This study focused on EPRAP, a novel EP4 receptor–associated protein, implicated in its signaling pathway. The generation of EPRAP-gene mutated mice permitted exploration of EPRAP functions  PGE2 receptors (EPs) mediate the effects of this molecule and include four subtypes: EP1–4  were generated as follows. The CD68–EPRAP/Fem1a transgene consists of the murine EPRAP/Fem1a cDNA downstream of the mouse CD68 promoter, which was derived from pDRIVE-mCD68 . We microinjected the transgene into fertilized C57BL/6 mouse eggs, and three lines were established from six founders. PCR, using a set of two primers , validatFor DSS-induced colitis, 8–10-week-old male WT, KO, and TG mice were given 2.5% (w/v) DSS  in their drinking water for 5 days, followed by a recovery period with regular drinking water through the end of the experiment (day 21). Control mice received DSS-free drinking water. For AOM/DSS-induced inflammatory tumorigenesis, 8–10-week-old male WT, KO, and TG mice received intraperitoneal injection of 12 mg/kg AOM  before three cycles of DSS administration as described above. All mice were maintained on food and water ad libitum, and were age-matched as well as co-housed for all experiments.ad libitum for 7 days [Eight- to ten-week-old male WT and EPRAP-deficient (KO) mice were each divided into two groups, EP4 agonist–treated and vehicle-treated. All four groups of mice  received 2.5% (w/v) DSS in their drinking water 6 bone marrow cells derived from femurs and tibias of adult WT or KO mice. Quantitative PCR verified peripheral blood chimerism: circulating blood cells were isolated and EPRAP mRNA expression was determined (see RNA isolation and quantitative PCR).Four- to eight-week-old male C57BL/6 wild type (WT) or EPRAP-deficient (KO) recipient mice were irradiated with X-rays (10 Gy) and injected intravenously with 6–14×10Rabbit polyclonal antibody against mouse EPRAP was raised by immunization with keyhole limpet hemocyanin–conjugated synthetic peptides corresponding to amino-acid residues 244–260, 330–346, and 629–645 of murine EPRAP. For immunohistochemistry of mouse samples, paraffin-embedded sections were routinely stained with one of the following antibodies: rat anti-F4/80 , rat anti–Gr-1 , rat anti-B220 (eBioscience), rat anti-CD4 (eBioscience), rat anti-CD8 (Abcam), hamster anti-CD11c (eBioscience), mouse anti-NCAM (Abcam), rabbit anti–phospho-NF-κB p105 (Ser933) , rabbit anti–phospho-MEK1/2 (Ser221) , or rabbit anti–phospho-p44/42 MAPK (Thr202/Tyr204) , or rabbit anti-EPRAP.For immunohistochemistry of human samples, paraffin-embedded sections were stained with rat anti-CD68 (Abcam), goat anti-FEM1A (Abcam), and rabbit anti–phospho-NF-κB p105 (Ser933), rabbit anti–phospho-MEK1/2 (Ser221), or rabbit anti–phospho-p44/42 MAPK (Thr202/Tyr204) antibodies. Staining with non-immune rat or rabbit IgG served as a negative control for each experiment.A validated scoring scheme assessed colitis. In brief, histological scoring was based on three parameters. Inflammation severity was scored as follows: 0, none; 1, mild; 2, moderate; 3, severe. Inflammation extent was scored as follows: 0, none; 1, mucosa; 2, mucosa and submucosa; 3, transmural. Crypt damage was scored as follows: 0, none; 1, basal 1/3 damaged; 2, basal 2/3 damaged; 3, crypts lost, surface epithelium present; 4, crypts and surface epithelium lost. Inflammation severity score, inflammation extent score, and crypt damage score were each multiplied by percent involvement , and the resultant products were summed to yield a histological score ranging from 0 to 40.Disease severity was determined based on the guidelines for the management of ulcerative colitis in Japan (modification of Truelove and Witts’ criteria) proposed by the Research Committee of Inflammatory Bowel Disease . The sevTo detect apoptotic cells, paraffin-embedded sections were stained using the ApopTag in situ apoptosis detection kit . Five different areas per tissue section were analyzed using BZ-H2C .5 cells were seeded into individual wells of a 96-well dish. After 10-minute treatment with different concentration of ONO-AE1-329 or vehicle, intracellular cAMP levels were determined using the Cyclic AMP EIA Kit .Peritoneal macrophages or colonic epithelial cells were harvested from 8-week-old WT, KO, and CD68–mEPRAP transgenic (TG) mice, and 3×10Colonic tissues were removed and homogenized in 1% NP-40 supplemented with protease inhibitor cocktail and PhosSTOP . The BD CBA assay  measured quantitative determinations of TNF-α, IL-6, and MCP-1 concentrations in the supernatants of tissue homogenates. Quantikine ELISA Immunoassay  measured IL-1β and CXCL1 concentrations.The Myeloperoxidase (MPO) Activity Colorimetric Assay Kit  measured MPO activity. MPO activity was compensated by the protein concentration of lysates.Lysates of colonic tissue extracts from DSS-treated WT and KO mice were assayed at a protein of 0.5 mg/ml or 0.05 mg/ml with the PathScan Signaling Nodes Multi-Target Sandwich ELISA Kit  according to the manufacture’s protocol.6 cells were seeded into individual wells of 48-well dishes. After 1 hour of treatment with 1 μg/ml of lipopolysaccharides   or vehicle, endogenous levels of phospho-MEK and phospho-ERK were determined at a protein of 0.3 mg/ml using the PathScan Phospho-MEK1 (Ser217/221) Chemiluminescent Sandwich ELISA Kit and the PathScan Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit  according to the manufacture’s protocol. Immunoblot analyses were also performed using the whole cell lysates of WT and KO peritoneal macrophages. Antibodies against phospho-NF-κB p105 (Ser933), phospho-MEK1/2 (Ser217/221), MEK1/2, phospho-p44/42 MAPK (Thr202/Tyr204), p44/42 MAPK (Erk1/2) were from Cell Signaling Technology: anti-mouse p105/p50 antibody was from Abcom. For quantitative PCR, peritoneal macrophages were treated with or without LPS, followed by total RNA isolation as described in the Materials and Methods. To test the effects of pharmacologic inhibition of MEK, cells were pretreated with U0126  at 10 μM for 1 hour prior to LPS treatment.Peritoneal macrophages were harvested from 10–12-week-old WT and KO mice and 1.5×10+CD11b+ macrophages were isolated using a FACSAria II flow cytometer (BD Biosciences), following staining of cell suspensions with FITC-labeled anti-CD45 (eBioscience) and PerCP–Cy5.5-labeled anti-CD11b (eBioscience). For analyses of the levels of phosphorylated forms of p105, MEK and ERK, freshly isolated macrophages were fixed in 4% paraformaldehyde/PBS, permeabilized in 0.5% Triton X-100/PBS, and stained with rabbit anti–phospho-p105, anti–phospho-MEK1/2, or anti–phospho-p44/42 MAPK antibody, followed by incubation with PE-conjugated donkey anti-rabbit IgG (eBioscience). Flow cytometry was performed using FACSAria II and analyzed using the FlowJo software .Lamina propria mononuclear cells and colonic epithelial cells were isolated as previously described . BrieflyTrizol Reagent (Invitrogen) was used to extract total RNA, Superscript III (Invitrogen) was used to synthesize single-stranded cDNA. The mRNA level for each target gene was determined by SYBR Green–based quantitative PCR using a LightCycler 480 system (Roche). Primer sequences are shown in To knockdown endogenous EPRAP expression in colon cancer cells , we used the BLOCK–iTPol II miR RNAi Expression Vector Kit with EmGFP (Invitrogen). miRNA-expressing plasmids were constructed according to the manufacturer’s protocol. Target sequences were as follows: miR–EPRAP #1, 5′-ACCAACCGAAAGCTATGCAAG-3′; miR–EPRAP #2, 5′-AAACCAACCGAAAGCTATGCA-3′. We used the pcDNA6.2/EmGFP–miR-negative control vector (miR–NC from Invitrogen) as a negative control. Plasmid transfection experiments were performed using Lipofectamine 2000 reagent (Invitrogen).3 cells were seeded in 96–well plates and cultured in the growth medium containing 10% fetal bovine serum for 0 h, 48 h, or 72 h. Then, absorbance at 492 nm was measured according to the manufacture’s protocol. All experiments were performed in octuplicate, and the results are shown as mean ± SEM of values.Following over expression  or knockt test or one-way ANOVA followed by Tukey-Kramer analysis. P values less than 0.05 were considered statistically significant.Data are presented as means ± SEM. Statistical comparisons between groups were made using Student’s S1 Fig(A) Targeting strategy for the generation of the EPRAP-deficient strain. (B) Genotype mapping of WT (+/+), heterozygous (+/-), and homozygous mutant (-/-) mice by genomic Southern blot analysis. Digestion of genomic DNA with AseI produced an 18-kb fragment from the WT allele, and a 10-kb fragment from the mutant allele, of the Eprap/Fem1a gene. (C) The EPRAP mRNA levels of WT (+/+), heterozygous (+/-), and homozygous (-/-) mutant mice in normal colonic extracts were analyzed by quantitative PCR (n = 3–4 each). Data represent fold induction of mRNA expression compared with WT (+/+). (D) A specific antibody against murine EPRAP was generated. EPRAP was immunostained with this antibody, using rectal sections obtained from DSS-treated WT (+/+) and homozygous EPRAP-deficient (-/-) mice.(TIF)Click here for additional data file.S2 Fig(A) Immunohistochemical staining to detect F4/80, Gr-1, B220, CD4, and CD8 in rectal sections of DSS-treated WT and EPRAP-deficient (KO) mice. (B) The mRNA levels of iNOS and CXCL10 in colonic stromal macrophages of DSS-treated WT and KO mice . Data represent fold induction of mRNA expression compared with WT.(TIF)Click here for additional data file.S3 Fig(A) H & E staining in rectal sections (non-polyp lesion) of AOM/DSS-treated WT and KO mice. (B) Immunohistochemical staining to detect F4/80, Gr-1, B220, CD4, and CD8 in rectal sections (non-polyp lesion) of AOM/DSS-treated WT and KO mice. (C) Histological colitis score in rectal sections (non-polyp lesion) of AOM/DSS-treated WT and KO mice . (D) The numbers of F4/80-, Gr-1-, B220-, CD4-, and CD8-positive cells infiltrated in colonic tissues per high-power field (400× magnification) in rectal sections (non-polyp lesion) of AOM/DSS-treated WT and KO mice . All values represent means ± SEM. *P < 0.05, **P < 0.01 vs. WT mice. Scale bars: 100 μm.(TIF)Click here for additional data file.S4 Fig DLD-1 cells were transiently transfected with V5-tagged human EPRAP expression construct or mock plasmid. Immunoblot analysis showing the enforced expression of recombinant EPRAP (A): quantitative real-time PCR showing increased levels of total cellular EPRAP mRNA (n = 4 each). **P < 0.01 vs. mock (B). (C) EPRAP gene knockdown experiments were performed as described in the Materials and Methods. Testing the efficiency of the gene silencing entailed quantitative real time PCR analysis (n = 4 each). *P < 0.05 vs. negative control.(TIF)Click here for additional data file.S5 Fig(A) Immunohistochemical detection of EPRAP-expressing cells. The colonic tissues were obtained from WT mice subjected to no treatment , DSS treatment, or AOM/DSS treatment. Scale bars: 100 μm. (B) Double–color immunofluorescence staining of rectal sections from control-, DSS-, or AOM/DSS-treated WT mice was performed with a combination of anti-EPRAP (red) and anti-F4/80 (green) antibodies. Scale bars: 20 μm. (C) Double–color immunofluorescence staining of rectal sections from DSS-treated WT mice was performed using a combination of anti-EPRAP (red) and anti-CD4, anti-CD8, anti-CD11c, anti-B220, or anti-NCAM (green) antibodies. Scale bars: 20 μm. (D) Double–color immunofluorescence staining of rectal sections from DSS-treated WT mice was performed using a combination of anti-EPRAP (red) and anti–Gr-1 (green) antibodies. Scale bars: 20 μm. (E) MPO activity was measured in protein extracts from colonic tissues from DSS-treated WT and EPRAP-deficient (KO) mice . MPO activity (left) and MPO activity compensated by the numbers of Gr-1–positive cells infiltrated in colonic tissues per high-power field ((F) The mRNA levels of TNF-α, IL-1β, CXCL1, and MCP-1 in colonic stromal macrophages of DSS-treated WT and EPRAP-deficient (KO) mice . (G) The EPRAP mRNA levels of lamina propria macrophages (LPMC) and epithelial cells isolated from the colonic tissues of WT mice with DSS treatment. Data represent fold induction of mRNA expression compared with WT. *P < 0.05, **P < 0.01 vs. WT mice.er field  (right).(TIF)Click here for additional data file.S6 Fig(A) Double-color immunofluorescence staining of rectal sections from DSS-treated WT and EPRAP deficient mice with or without EP4 agonist treatment (as described in the (B) The percentages of phospho-MEK (left) or phospho-ERK (right) positive cells in F4/80 positive macrophages (n = 5 each).(TIF)Click here for additional data file.S7 Fig(A) Immunoblot analyses were performed to examine the levels of phosphorylated forms of p105, MEK and ERK. Figures are representative of three independent experiments. (B) The expression levels of phosphorylated forms of MEK and ERK were examined (n = 4 each). (C) The mRNA levels of TNF-α, IL-1β, IL-6, and CXCL1 in peritoneal macrophages of WT or KO mice (n = 7–9 each). (D) The mRNA levels of TNF-α, IL-1β, IL-6, and CXCL1 in peritoneal macrophages of WT or KO mice pre-treated with MEK inhibitor (U0126) (n = 6 each). Data represent fold induction of mRNA expression of LPS-treated cells compared with that of vehicle-treated cells. *P < 0.05, **P < 0.01 vs. WT mice.Peritoneal macrophages were isolated from 10–12-week-old of WT or KO mice. Cells were incubated with LPS or vehicle for one hour, followed by whole cell extraction. (TIF)Click here for additional data file.S8 Fig(A) Double–color immunofluorescence staining of rectal sections from WT and CD68–mEPRAP transgenic (TG) mice was performed with a combination of anti-EPRAP (red) and anti-F4/80 (green) antibodies. (B) The mRNA levels of EPRAP in CD68-positive bone marrow cells (n = 4 each). Data represent fold induction of mRNA expression compared with WT. **P < 0.01 vs. WT mice.(TIF)Click here for additional data file.S9 Fig(A) Survival curves during the DSS treatment. WT and TG mice had no significant difference in mortality . (B) Percent changes in body weight . WT and TG mice had no significant difference. (C) Immunohistochemical staining to detect F4/80, Gr-1, B220, CD4, and CD8 in rectal sections of DSS-treated WT and TG mice.(TIF)Click here for additional data file.S10 Fig(A) H & E staining in rectal sections (non-polyp lesion) of AOM/DSS-treated WT and TG mice. (B) Immunohistochemical staining to detect F4/80, Gr-1, B220, CD4, and CD8 in rectal sections (non-polyp lesion) of AOM/DSS-treated WT and TG mice. (C) Histological colitis score in rectal sections (non-polyp lesion) of AOM/DSS-treated WT and KO mice . (D) The numbers of F4/80-, Gr-1–, B220-, CD4-, and CD8-positive cells infiltrated in colonic tissues per high-power field (400× magnification) in rectal sections (non-polyp lesion) of AOM/DSS-treated WT and TG mice . All values represent means ± SEM. **P < 0.01 vs. WT mice. Scale bars: 100 μm.(TIF)Click here for additional data file.S11 Fig(A) Double–color immunofluorescence staining of colonic sections of UC patients was performed with a combination of anti-EPRAP (green) and anti–phospho-p105 , anti–phospho-MEK , or anti–phospho-ERK  antibodies. Scale bars: 20 μm.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


While the immune system has the capacity to recognize and destroy melanoma, tolerance mechanisms often hinder the development of effective anti-tumor immune responses. Since many melanoma antigens are self proteins expressed in normal melanocytes, self antigen exposure before tumor development can negatively impact the function of T cells specific for these self/tumor antigens. However, the contribution of self tolerance to anti-melanoma T cell dysfunction remains largely unexplored. We have previously described a TCR transgenic (Tg) mouse model in which T cells specific for the self/melanoma antigen, tyrosinase-related protein 1 (TRP1), develop in the presence of endogenous TRP1 expression (Ag+) and diminished antigen presentation due to the absence of gamma-interferon-inducible lysosomal thiol reductase (GILT-/-). We show that TRP1-specific T cells from these Ag+GILT-/-Tg mice do not protect from melanoma tumor growth, fail to induce autoimmune vitiligo, and undergo diminished proliferation compared to T cells from Ag-GILT+/+Tg mice. Despite an increased frequency of TRP1-specific Treg cells in Ag+GILT-/-Tg mice compared to Ag-GILT+/+Tg animals, Treg cell depletion only partially rescues the proliferative capacity of T cells from TRP1-expressing mice, suggesting the involvement of additional suppressive mechanisms. An increased percentage of melanoma-specific T cells from Ag+GILT-/-Tg animals express PD-1, an inhibitory receptor associated with the maintenance of T cell exhaustion. Antibody blockade of PD-1 partially improves the ability of TRP1-specific T cells from Ag+GILT-/-Tg mice to produce IL-2. These findings demonstrate that melanoma-specific T cells exposed to a self/melanoma antigen in healthy tissue develop an exhaustion-like phenotype characterized by PD-1-mediated immunosuppression prior to encounter with tumor. The immune system is capable of recognizing melanoma tumors, and patients readily develop melanoma-specific T cell responses , 4, 5, 6Since many of the known melanoma antigens are self proteins expressed in normal melanocytes, it is important to determine the role of self antigen exposure in melanoma-specific T cell dysfunction. Human studies of tumor-infiltrating lymphocytes specific for self/melanoma antigens are unable to assess the impact of self antigen exposure prior to tumor development on T cell tolerance , 17, 18.Our laboratory has developed a mouse model to study mechanisms that constrain CD4+ T cell-mediated immunity to melanoma antigens that are also self antigens , using tOur laboratory has previously shown that TRP1-specific T cells from Ag+GILT-/-Tg mice fail to induce vitiligo after adoptive transfer in part due to a four-fold increase in the percentage of TRP1-specific Foxp3+ Treg cells in comparison to Ag-GILT+/+Tg mice . While TTo test the anti-melanoma activity of T cells developing in Ag+GILT-/-Tg mice in comparison to Ag-GILT+/+Tg mice and Ag+GILT+/+Tg mice, animals were subcutaneously injected with B16 melanoma cells and followed for tumor growth. The anti-tumor activity of TRP1-specific T cells from Ag-GILT+/+Tg mice has been described previously , 21. ConWe have previously shown that GILT-deficient antigen presenting cells (APCs) are diminished in their ability to activate TRP1-specific T cells due to reduced presentation of TRP1 . TherefoConversely, adoptive transfer of TRP1-specific T cells from Ag+GILT-/-Tg mice failed to induce vitiligo in Ag-expressing RAG1-/- mice with or without GILT . FurtherTRP1-specific T cells from Ag+GILT-/-Tg mice were recovered in reduced percentages after adoptive transfer in comparison to T cells from Ag-GILT+/+Tg mice, suggesting that T cells from Ag+GILT-/-Tg mice may have impaired antigen-induced proliferation. Therefore, we compared the ability of CD4+ T cells from these two strains to proliferate in response to TRP1. CD4+ T cells from Ag+GILT-/-Tg mice demonstrated markedly diminished TRP1-specific proliferation in comparison to T cells from Ag-GILT+/+Tg animals across a range of peptide concentrations p<0.001; . In addiWe have previously shown that Foxp3+ Treg cells contribute to TRP1-specific T cell tolerance in the Ag+GILT-/-Tg mouse model . To deteTRP1-specific T cells from Ag+GILT-/-Tg mice display many signs of T cell exhaustion including an antigen-experienced phenotype, impaired cytokine production , and dimTo determine if PD-1 inhibits IL-2 production by TRP1-specific T cells, we performed blocking experiments using a monoclonal antibody to PD-1. IL-2 production by CD4+ T cells from Ag+GILT-/-Tg mice was diminished in comparison to T cells from Ag-GILT+/+Tg animals p<0.001; , consistBy crossing the TRP1-specific TCR transgenic mouse strain  onto thein vivo thymidine was added for the final 18 hours of culture.CD4+ TRP1-specific T cells (1x105) were cocultured for 48 hours with 5x105 T cell-depleted wild-type spleen cells and murine TRP1109-130 peptide (10 μg/mL) or BSA (1mg/ml) with either PD-1 blocking antibody (5μg/ml) or isotype control antibody (5μg/ml). The IL-2 concentration in culture supernatants was determined by ELISA (BD Biosciences).Positively selected CD4+ TRP1-specific T cells (1x10


Ex vivo analyses revealed that T cells in the skin were the primary IL-10-producing cells. To understand the distribution of IL-10-producing T cells in vivo, we performed multiphoton intravital microscopy (MPM) of VV-infected mice, assessing the location and dynamic behavior of IL-10 producing cells. Although virus-specific T cells were distributed throughout areas of the inflamed skin lacking overt virus-infection, IL-10+ cells closely associated with large keratinocytic foci of virus replication where they exhibited similar motility patterns to bulk antigen-specific CD8+ T cells. Paradoxically, neutralizing secreted IL-10 in vivo with an anti-IL-10 antibody increased viral lesion size and viral replication. Additional analyses demonstrated that IL-10 antibody administration decreased recruitment of CCR2+ inflammatory monocytes, which were important for reducing viral burden in the infected skin. Based upon these findings, we conclude that spatially concentrated IL-10 production limits cutaneous viral replication and dissemination, likely through modulation of the innate immune repertoire at the site of viral growth.Skin infection with the poxvirus vaccinia (VV) elicits a powerful, inflammatory cellular response that clears virus infection in a coordinated, spatially organized manner. Given the high concentration of pro-inflammatory effectors at areas of viral infection, it is unclear how tissue pathology is limited while virus-infected cells are being eliminated. To better understand the spatial dynamics of the anti-inflammatory response to a cutaneous viral infection, we first screened cytokine mRNA expression levels after epicutaneous (ec.) VV infection and found a large increase the anti-inflammatory cytokine IL-10.  While ineffective antiviral immune responses can result in illness or even death, excessive host responses can also cause substantial injury. Anti-inflammatory proteins play an important regulatory role in limiting immune-mediated damage, but it is unknown where the cells making these modulators need to be for the greatest effect. The best-described immune-response-limiting protein is the cytokine interleukin-10 (IL-10), which is produced during infections with disparate pathogens including viruses, bacteria, and parasites. Despite the preponderance of IL-10 production during infection, we do not know the tissue distribution of this cytokine or whether it acts in localized areas. To address these questions, we studied the behavior of IL-10-producing cells after infecting mice with vaccinia virus (VV), the attenuated vaccine virus used to eradicate smallpox. Using microscopy to image infection in the skin of living mice, we found that IL-10 is produced by cells that surround areas of virus replication, rather than throughout the tissue as we anticipated. Even more surprisingly, we found that IL-10 promotes (rather than impedes) virus clearance, likely by shaping the tissue response of innate immune cells. These data show that supplying anti-inflammatory cytokines at precise areas of infected tissue can dramatically limit viral replication and damage. For many viruses, however, the immune response extends beyond that necessary for viral clearance and creates disease symptoms. For example, infection with certain influenza virus (IAV) strains results in lung recruitment of high numbers of neutrophils (and other leukocytes) leading to a fast-progressing viral pneumonia and extensive lung damage , 2. Neutchildren . Furtherchildren . Thus, tIL-10 is an important anti-inflammatory cytokine that quells innate and adaptive immune responses during both infection and autoimmunity –9. Origi+ T cells can also produce IL-10 and even represent a major source of IL-10 in infected organs [+ T cells appears to be dictated by both viral tropism and the site of infection, but the principle function ascribed to these cells is suppression of inflammatory tissue damage rather than direct alteration of viral replication. For example: antibody (Ab) blockade of the IL-10 receptor (IL-10R) during IAV infection increases lung inflammation and mortality without reducing viral burden [+ CD8+ effector T cells prevent RSV-induced inflammatory lung pathology [+ CD8+ T cells limit CNS demyelination and slightly increase viral burden as a necessary cost [Paradoxically, during some acute viral infections highly activated, pro-inflammatory CD8d organs –24. The l burden . Similarathology . During ary cost . The extin situ or the spatiotemporal contribution of IL-10-producing cells to viral clearance is unknown. Previously, we reported that during vaccinia virus (VV) infection of the skin, myeloid innate immune cells and CD8+ effector T cells exhibited strikingly different distributions [Multiple aspects of IL-10 production have been proposed to shape disease outcome, including IL-10 production kinetics and shifts in the anatomical location of IL-10 depots , 25. Theibutions . Thus, tin situ after epicutaneous (ec.) VV infection. VV skin infection induces a large population of IL-10+ CD4+ and IL-10+ CD8+ T cells in the infected tissue. Intriguingly, IL-10+ T cells more closely localize to viral replicative foci than do the bulk population of antiviral CD8+ T cells in the tissue. Further, IL-10+ T cells move in paths adjacent to areas of virus infection, and neutralization of IL-10 enhances virus spread and shapes the innate immune population located adjacent to these foci. Together, our data demonstrate that spatially directed anti-inflammatory cytokine production inhibits viral dissemination within the infected tissue and illuminate a novel aspect of antiviral immunity.Here, we analyze the dynamics of IL-10-producing cells + T cells and innate Ly6G+ cells, synergize to control recombinant VV (rVV) infection of the skin [1-polarized cellular infiltrate, viral lesions after epicutaneous (ec.) infection of C57Bl/6 mice resolve in 14 days with limited, rapidly repaired tissue damage.Multiple inflammatory immune effectors, including cytotoxic CD8the skin . DespiteTo understand the potential mechanisms of immune self-restraint during ec. VV infection, we performed qPCR arrays, analyzing expression of cytokine mRNAs isolated from infected tissue at 6.5 days post-infection (d.p.i.) . At thisten IRES GFP-enhanced reporter mice)) [+ IL-10-producing cells in the skin by 5 d.p.i., peaking on day 6 at 2.2% of total cells, and then declining to a near constant level of ~1% of total cells until 10 d.p.i.  (+ cells (1.84%) using an alternative IL-10 reporter mouse )   . We foun+ skin cells on day 6 post-recombinant vaccinia virus (rVV)-infection of TIGER mice were CD8+ T cells (+ T cells comprised the second largest GFP+ population (~15%) on day 6, with minimal contributions from dendritic cells  or macrophages (CD11b+CD11c-). GFP+ populations were virtually identical in Vert-X reporter mice  of IL-10gfp T cells , as defis or DCs . Over tinfection . We confstaining .+ CD8+ T cells being more numerous.Together, these data show that ec. VV infection elicits a sizeable population of IL-10-expressing T cells in the skin over the course of acute infection, with IL-106 plaque forming units (PFU)/ear and dropped to close to the limit of detection by 10 d.p.i. Using flow cytometry, we enumerated total CD4+ and CD8+ T cells in ear single cell suspensions over the course of infection .We next examined VV replication after ec. infection of the skin . Viral tnfection . Few T cells/ear ). Notabl+ T cells were restricted to the infected tissue, we analyzed the spleen and draining lymph nodes (dLNs) from VV-infected animals  was present in DLNs, likely representing regulatory T cells [To determine whether IL-10 animals . We dete T cells  Fig 2E)+ T cellsThus, at peak, between 10 and 15% of T cells express IL-10 in the skin during VV infection.+ CD8+ T cells responding to IAV, RSV, or coronavirus infection co-produce a number of pro-inflammatory effector cytokines [+ T cells expressed T-bet  , reker CD69 . In both0+ cells . We did  the ear . Thus, b+ cells in VV-infected reporter (TIGER) mice. Consistent with ex vivo experiments, we detected few IL-10gfp+ cells (green) in uninfected skin  [+ cells (green) surrounding keratinocytic foci of viral replication (pseudocolored magenta)   )  ), we con+ cells were recruited around VV replicative foci   . Near le lesions . Intriguustering  , we next used MPM imaging to compare the distribution of IL-10gfp+ cells to adoptively transferred virus-specific T cells endogenously activated in IL-10gfp-reporter mice (+ dsRed+ OT-I CD8+ T cells (antigen specificity = Kb-SIINFEKL) into naïve IL-10gfp reporter mice, then infected ec. On day 6 p.i. (rVV expression of BFP is pseudocolored magenta), we detected both IL-10gfp+ cells (green) and IL-10gfp-/- adoptively transferred OT-I CD8+ T cells (red) at the border of viral lesions   were present in the same area  . Furtherame area . Thus, u+ cells in relation to dsRed+ OT-I CD8+ T cells within the same imaging field  engaged and motionless, 2) motile at the perimeter of infection, or 3) only transiently associated with the borders of lesions. To examine these possibilities, we analyzed the movement of peri-lesional IL-10gfpng field . Intrigusessions  Movies. nfection . We did + cells moved at slightly lower mean speeds than dsRed+ OT-I CD8+ T cells (mean speed of ~2.3 vs. 2.8 μm/min) (+ cells were motile (defined by a speed > 2.5 μm/min)  , though  μm/min) . The bi- μm/min) .+ cells are mobile in confined areas around keratinocytic foci of infection.Together, these data show that most IL-10gfp-/- animals). On day 7 p.i., we detected a significantly decreased number and percentage of CCR2+ tissue monocytes in the ears of IL-10-neutralized animals  [+ cells . In contrast, VV infection resulted in dramatic accumulation of CCR2rfp+ cells in areas with viral-driven GFP expression   Fig 7B)+ tissue [+ cells . In uninpression . Many ofpression . Furthercontrols .+ monocytes, which localize to areas of viral infection and reduce viral titers in the skin.Together, these data suggest that locally produced IL-10 enhances the recruitment of CCR22 mosaic) image increased 2.3-fold (4.2 to 9.6) in IL-10-neutralized ears (5 to 5.7 x 105 VV genomes/μg DNA) after IL-10 neutralization , and alinfected . Interes+ monocytes. Administration of IL-10 neutralizing Ab resulted in an approximately 25% reduction in the number of CCR2+ monocytes in the skin  macrophages promote tissue restoration post-trauma, infection, or allergic inflammation [e.g. in individuals with atopic dermatitis [et al. recently used tape stripping and ovalbumin-sensitization to create atopic dermatitis-like skin lesions and then infected mice with VV [17 responses.IL-10 could participate in restriction of VV replication in the skin via other mechanisms besides CCR2ammation –48. Becarmatitis , 50), IL with VV . IL-10 k-/- mice or Ab-mediated IL-10R blockade decreased ovarian VV titers slightly at 7 d.p.i. [Additionally, IL-10 may directly inhibit VV replication by inducing innate anti-viral pathways in infected cells. An early study characterizing an IL-10-overexpressing rVV revealed no effect on viral replication, however IL-10 may already be saturating in wild-type mice . Intrape7 d.p.i. , 54. Byr7 d.p.i. , 56) dif7 d.p.i. . VV repl+ monocytes, our findings demonstrate that the effects of IL-10 are likely to be highly dependent on the location of IL-10 secreting T cells. Interestingly, nearly all IL-10-producing cells in infected reporter mice were amassed at the borders of viral lesions, rather than being more distributed throughout the inflamed, edematous tissue as were many virus-specific T cells -Il10tm1.1Karp/J stock #14530, “Vert-X) and bred in house with B6(Cg)-Tyrc-2J/J  for heterozygosity and homozygosity. 6–12-wk-old adult mice were used in all experiments. B6(Cg)-Tyrc-2J/J  were also used where indicated. All mice were housed under specific pathogen–free conditions  and maintained on standard rodent chow and water supplied ad libitum.Specific pathogen-free C57BL/6, B6(Cg)-Tyrc-2J/J, and IL-10gfp reporter transgenic mice on a C57BL/6 background were acquired from The Jackson Laboratory . CeAll animal experiments were conducted in accordance with the Animal Welfare Act and the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. NIAID animal facilities have full accreditation from the Association for Assessment and Accreditation of Laboratory Animal Care and are PHS-assured (Assurance Number: # A4149-01). All animal procedures were approved by the NIAID Animal Care and Use Committee.8 PFU/mL rVV stock placed on the ear skin and gently poked a total of 10 times per ear with a bifurcated needle (similar to the human vaccination protocol). VVs used in this study were VV-Ova , VV-NP-S-GFP (with the influenza nucleoprotein (NP), SIINFEKL peptide (S), and green fluorescent protein; fluorescence is nuclear), VV-NP-S-BFP, and VV-BFP-Ub-SIINFEKL (cellular ubiquitin hydrolase liberates SIINFEKL peptide from the fusion protein). All have been previously described [Mice were infected ec. in the ear pinnae by using a drop of 1x10escribed  and viru+ T cells were defined as CD8α+, CD8β+ cells (to exclude CD8α+ dendritic cells).Single cell suspensions of ears, cervical lymph nodes and spleens were prepared by collagenase digestion to liberate immune cells  for 1 hr at 37°C and processed into single cell suspensions prior to filtration through 70 μm nylon cell strainers. The following antibodies were used to stain the cells: CD45 (clone 30-F11), CD8a (clone 53–6.7), CD8b (clone eBioH35-17.2), CD4 (clone RM4-5), CD11c (clone HL3), CD11b (clone M1/70), CD69 (clone H1.2F3), IL-10 (clone JES5-16E3), IFN-γ (clone XMG1.2), Granzyme B (clone NGZB), T-bet (clone eBio4B10). Antibodies were purchased from eBiosciences and BD Biosciences. CD8For intracellular cytokine staining, brefeldin A  was added during collagenase digestion, and liberated cells were incubated an additional 3 hours in BFA-containing RPMI (Life Technologies) at 37°C. Cells were stained with 5 μg/mL of ethidium monoazide (EMA) for 15 min in the dark, exposed to light for 10 min to mark dead and dying cells. Cells were subsequently stained for the cell surface markers CD4, CD8, CD69, and CD45, fixed at room temperature for 30 min with eBiosciences FoxP3 Fix/Perm buffer, washed, then stained with anti-IFN-γ, anti-TNF-α, anti-Granzyme B, anti-T-bet and/or anti-FoxP3 in Perm buffer overnight at 4°C. Cells were analyzed on an LSR II flow cytometer (BD Biosciences) and resultant data analyzed using FlowJo (Treestar).Unless otherwise indicated, mice received 0.25 mg of anti-IL-10  intraperitoneally (i.p.) on day 5 and day 6 post infection.-/- cells. Cells were incubated for 2 days before counting the resulting plaques.Ears were removed at various times p.i., collagenase digested for 1 hour at 37°C, and disrupted by vigorous pipetting. This suspension was freeze-thawed 3X, sonicated 3X, serially diluted and plated on TK2 first strand kit (Qiagen). Common cytokine PCR array (Qiagen) was used to quantitate cytokine mRNA fold increase over uninfected controls, according to the manufacturer’s instructions.Total RNA was isolated from whole ears using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was generated using RT2 SYBR green qPCR Master Mix (SABiosciences). To quantify VV genome copies, a standard curve was generated using DNA from purified VV stock with a known PFU (determined by plaque assay). Viral copies were determined using a standard curve and were normalized to the amount of total input DNA.DNA was isolated from whole ears using the ArchivePure DNA Tissue Kit,  according to the manufacturer’s guidelines. Viral genomes were quantified by real-time PCR using primers specific for VV E3L gene. Primer sequences: 5′-GCAGAGATTGTGTGTGCGGCTATT-3′ and 5′-GGTGACAGGGTTAGCATCTTTCCA-3′ as previously reported . Amplifiz-step for a total depth of 150 μm for 1 min intervals between series (needed to switch lasers). For 3D reconstructions, images were acquired at 1x zoom using 1024x1024 resolution and 1 μm z-steps for a total depth of 80–300 μms.MPM imaging was performed essentially as described previously , 62, 63.Ears were fixed in PLP fixative  overnight as reported in , cryopro2 imaged area and individual fields (tiles) were merged into one image. Total image fluorescence was calculated using ImageJ software.Scans were taken of entire infected ears equaling a 7 mmFor MPM analysis, maximum intensity projections (MIPs) were processed from z-stacks using Imaris (Bitplane). Because both collagen (second harmonic generation) and BFP appear in the blue channel, BFP expression was pseudocolored magenta. For tracking cellular movement, images were processed using a Gaussian filter, tracks were calculated using the “spot” function of Imaris. Average speeds were calculated using the spot detection function and the following parameters: autoregressive motion, gapclose 1, 7.5 μm object diameter, 20 μm maximum distance. Following automated analyses, tracks were analyzed individually for erroneous connections. Track straightness was calculated according to the standard convention of displacement/track length.t test.Significances were calculated using GraphPad (Prism) using unpaired two-tailed Student’s S1 Movie+ cells and autofluorescent hairs = green. Collagen (dermis) = blue. Scale bars = μm. See also Three-dimensional rotation of a maximum intensity projection (MIP) of an MPM image of an IL-10gfp reporter mice (TIGER) 6 d.p.i. with VV NP-S-Blue fluorescent protein . IL-10gfp(MOV)Click here for additional data file.S2 Movie5 adoptively transferred dsRed+ OT-I CD8+ T cells (red) before infection.  Image was acquired 6 d.p.i. Virus-infected cells = pseudocolored magenta. IL-10gfp+ cells and autofluorescent hairs = green. Collagen (dermis) = blue. Scale bars = μm. See also Three-dimensional rotation of a maximum intensity projection (MIP) of an MPM image of an IL-10gfp-reporter mice (TIGER) that received 1 x 10(MOV)Click here for additional data file.S3 Movie5 adoptively transferred dsRed+ OT-I CD8+ T cells (red) before infection.  Image was acquired in an area that lacked virus-driven fluorescent protein expression (which would be magenta) on 6 d.p.i. IL-10gfp+ cells and autofluorescent hairs = green. Collagen (dermis) = blue. Scale bars = μm. See also Three-dimensional rotation of a maximum intensity projection (MIP) of an MPM image of an IL-10gfp-reporter mice (TIGER) that received 1 x 10(MOV)Click here for additional data file.S4 Movie5 adoptively transferred dsRed+ OT-I CD8+ T cells (red) before infection.  Image was acquired on 6 d.p.i. Virus-infected cells = pseudocolored magenta. IL-10gfp+ cells and autofluorescent hairs = green. Collagen (dermis) = blue. Scale bars = μm. Time = min. See also Movie of MIP images taken 6 d.p.i. with VV NP-S-BFP (pseudocolored magenta) over a 2 hr imaging session in an IL-10gfp-reporter mice (TIGER) that received 1 x 10(MOV)Click here for additional data file.S5 Movie5 adoptively transferred dsRed+ OT-I CD8+ T cells (red) before infection.  Image was acquired on 6 d.p.i. Virus-infected cells = pseudocolored magenta. IL-10gfp+ cells and autofluorescent hairs = green. Collagen (dermis) = blue. Scale bars = μm. Time = min. See also Movie of MIP images taken 6 d.p.i. with VV NP-S-BFP (pseudocolored magenta) over a 2 hr imaging session in an IL-10gfp-reporter mice (TIGER) that received 1 x 10(MOV)Click here for additional data file.S6 Movie+ cells and autofluorescent hairs = green. Collagen (dermis) = blue. Time = min. Scale bars = μm. See also Movie of MIP images taken 6 d.p.i. with VV-NP-S-BFP (pseudocolored magenta) over a 20 min. imaging session without adoptive transfer of other T cells. IL-10gfp(MOV)Click here for additional data file.S7 Movie+/- mouse cells (red CCR2+ monocytes). Image was acquired 7 d.p.i. Movement of one virus-infected monocytes is highlighted with a circle. Collagen (dermis) = blue. Scale bars = μm. Time = min. See also Movie of MIP images taken 6 d.p.i. with VV-NP-S-GFP (green) over an hr imaging session in an CCR2rfp(MOV)Click here for additional data file.S1 Fig+ T cells isolated from the skin producing IL-10 (determined by antibody staining for intracellular protein) on days 6, 12, and 13 post-infection with recombinant vaccinia virus expressing ovalbumin (Vac-Ova). White bars = cells analyzed directly ex vivo. Black bars = cells that were restimulated for 5 hours with Vac-Ova. Pseudocolored dot plots for an individual animal on day 6 post-infection are shown on the right. IL-10 staining is on the x-axis. B) as in A) except gating on CD8+ T cells. N = 3 mice/group. Error bars = SEM.A) Percentage of CD4(TIF)Click here for additional data file.S2 Fig+ cells, then on CD8+ T cells, then on CD8+ and FoxP3+ cells (stained intracellularly using a kit from eBioscience). Gate shows the percentage of FoxP3+ CD8+ T cells on day 6.Flow cytometric dot plot of single cell suspensions of ears 6 days post-VV-infection. Cells were gated on CD45(TIF)Click here for additional data file.S3 Fig+ cells over 20 min imaging periods. Dots represent individual cells; groups represent different experiments. Means of average speeds are shown with a black bar. B) IL-10gfp+ cell track straightness (track displacement/track length) C) Mean track length of IL-10gfp+ cells over 20 min. D) Percentage of IL-10gfp+ cells that were stopped or slowly motile, moving at average speeds less than 2.5 μm/min. Dots show the percentage of each of 8 experiments.A) Average cellular speeds of IL-10gfp(TIF)Click here for additional data file.S4 Fig+ leukocytes, then on CCR2+ CD11b+ cells. (note: CCR2 was examined by cell-surface antibody staining due to lack of rfp detection on our cytometer) B) Numbers of CCR2+ monocytes per ear on day 7 post-infection. We selected heterozygous mice for further analysis because of the reduction in number of monocytes (similar to the effect of IL-10 treatment).Monocyte populations in CCR2rfp null, heterozygous, and homozygous mice 7 days post-VV-infection. A) Flow cytometric plots of single cell suspensions generated from infected ears. Cells were gated on CD45(TIF)Click here for additional data file.S1 Data(DOCX)Click here for additional data file.


NK cells detect tumors through activating surface receptors, which bind self-antigens that are frequently expressed upon malignant transformation. To increase the recognition of tumor cells, the extracellular domains of ligands of the activating NK cell receptors NKp30, NKp80 and DNAM-1  were fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2), which is displayed by various solid tumors. The resulting immunoligands, designated B7-H6:HER2-scFv, AICL:HER2-scFv, and PVR:HER2-scFv, respectively, bound HER2 and the addressed NK cell receptor. However, whereas B7-H6:HER2-scFv and AICL:HER2-scFv triggered NK cells to kill HER2-positive breast cancer cells at nanomolar concentrations, PVR:HER2-scFv was not efficacious. Moreover, NK cell cytotoxicity was enhanced synergistically when B7-H6:HER2-scFv or AICL:HER2-scFv were applied in combination with another HER2-specific immunoligand engaging the stimulatory receptor NKG2D. In contrast, no improvements were achieved by combining B7-H6:HER2-scFv with AICL:HER2-scFv. Additionally, B7-H6:HER2-scFv and AICL:HER2-scFv enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) by the therapeutic antibodies trastuzumab and cetuximab synergistically, with B7-H6:HER2-scFv exhibiting a higher efficacy. In summary, antibody-derived proteins engaging NKp30 or NKp80 may represent attractive biologics to further enhance anti-tumor NK cell responses and may provide an innovative approach to sensitize tumor cells for antibody-based immunotherapy. Natural killer (NK) cells are innate immune cells playing a major role in the host's early defense against infections and tumors , 2. TheyNK cell surface receptors promoting natural cytotoxicity include natural killer group 2 member D , NKp30 (CD337), NKp44, CD336), NKp46 (CD335), NKp80 , DNAX accessory molecule 1  and others [, NKp46  as a target antigen on solid tumors  and to tThe extracellular domains of B7-H6, AICL and PVR were genetically fused to a HER2-specific scFv derived from the humanized antibody humAb4D5-8 . The resThe binding activities of the immunoligands were analyzed with HER2-positive human SK-BR-3 cells and flow cytometry Fig. . B7-H6:HThe cytotoxic activities of B7-H6:HER2-scFv, AICL:HER2-scFv and PVR:HER2-scFv were analyzed in chromium release assays Fig. . Human mTo assess the proposed specific mode of action of the immunoligands blocking experiments were performed, in which either the effector molecule or the HER2 target antigen were masked by antibodies or antibody-derivatives Fig. . Thus, lThe killing efficacy of the immunoligands was assayed at varying concentrations using MNC from several different donors. Both B7-H6:HER2-scFv and AICL:HER2-scFv triggered lysis of SK-BR-3 Fig.  and MDA-To demonstrate that the immunoligands triggered NK cells, NK cells from different donors were purified by negative selection by MACS technology and analyzed instead of MNC as effector cells for B7-H6:HER2-scFv and AICL:HER2-scFv in chromium release experiments Fig. . As expeBoth B7-H6:HER2-scFv and AICL:HER2-scFv are glycosylated proteins. To analyse the impact of glycosylation on cytotoxic effects mediated by the immunoligands, deglycosylated variants of both immunoligands were generated by enzymatic digestion under native conditions . Efficiehigh NK cell subpopulation, FcγRIIIa was found abundantly expressed with 70,000 molecules per cell, whereas NKp30, NKp80, DNAM-1 and TACTILE were expressed at significantly lower levels of between 1,000 (NKp30) and 5,000 (NKp80) molecules per cell. Interestingly, despite 5-fold lower expression levels of NKp30, B7-H6:HER2-scFv was more efficacious than AICL:HER2-scFv, suggesting that additional factors are important.Because the expression levels of NK cell receptors may influence the cytotoxic activity of the immunoligands, the expression of different NK cell receptors was analyzed by calibrated flow cytometry, and SABC per cell were determined as a measure of the cell surface numbers of the receptors Fig. . Hence, NK cells are able to integrate activating signals transmitted via different receptors . Thus, tADCC represents an important effector function for many therapeutic antibodies , 32. TheWith the aim to increase the visibility of tumor cells to NK cells, three bispecific immunoligands carrying a HER2-specific scFv and the ECD of B7-H6, AICL or PVR were generated to trigger NK cells via the activating receptors NKp30, NKp80 or DNAM-1, respectively. Whereas PVR:HER2-scFv did not mediate cytotoxic effects, both B7-H6:HER2-scFv and AICL:HER2-scFv promoted NK cell cytotoxicity and induced killing of HER2-positive tumor cells. B7-H6:HER2-scFv as well as AICL:HER2-scFv synergized with the immunoligand ULBP2:HER2-scFv engaging NKG2D and each were able to boost ADCC by monoclonal antibodies, suggesting that both NKp30 and NKp80 may represent attractive effector molecules on NK cells for the design of immune-modulatory biological agents.Raising NK cells against tumors may be a promising treatment option for various types of cancer . SeveralThe potency of individual immunoligands may reflect the characteristics of the corresponding NK cell receptor. Of note, the NK cell receptors bound by the immunoligands analyzed in this study differed in their intracellular signaling pathways. Whereas NKp30 similar to FcγRIIIa is coupled to the immunoreceptor tyrosine-based activation motif (ITAM)-bearing CD3ζ or FcεRIγ chains, NKp80, DNAM-1 and NKG2D function ITAM-independent , 6. ThusThe lack of activity of PVR:HER2-scFv may be attributed to a weak signal transmitted by engagement of the receptor, an unfavorable cell surface distribution of the HER2-bound immunoligand, or the strict requirement for engagement of additional activating or co-activating receptors. Moreover, also the recognition of additional NK cell receptors may play an important role. Whereas B7-H6, AICL and ULBP2 each ligate a single NK cell receptor, PVR is recognized not only by DNAM-1, but also by T cell activation increased late expression  and T cell Ig and ITIM domain (TIGIT), which both are expressed by NK cells as well. Whereas the adhesion molecule DNAM-1 promotes NK cell cytotoxicity , TIGIT pImportantly, synergistic effects were achieved when distinct immunoligands were combined to engage different NK cell receptors simultaneously. Of note, synergistic effects were achieved despite cross-competitive binding to the same HER2 epitope. However, not all combinations lead to increased tumor cell lysis and no significant improvements were obtained when B7-H6:HER2-scFv was combined with AICL:HER2-scFv. Thus, synergy was observed for the combination between B7-H6:HER2-scFv and ULBP2:HER2-scFv as well as between AICL-HER2-scFv and ULBP2:HER2-scFv. These findings indicate cooperation between NKG2D and NKp30 or NKp80 in triggering NK cell cytotoxicity and are in agreement with our previous results obtained with CD20-directed immunoligands containing either ULBP2 or B7-H6 . In variThe induction of synergistic effects by engagement of different NK cell receptors along with the possibility to target two different target antigens may even allow preferential killing of tumor cells, given that malignant cells express both target antigens at significant densities while healthy cells do not. In this setting, the selection of appropriate target antigens will be critical to realize this “dual-dual targeting” concept and further studies are warranted to provide proof-of-concept.In conclusion, B7-H6:HER2-scFv and AICL:HER2-scFv exerted cytotoxic abilities as single agents and in combination with NKG2D-specific immunoligands or monoclonal antibodies. In particular with their abilities to boost ADCC by therapeutic antibodies in a synergistic manner these immunoligands represent interesting novel immunomodulatory biologics designed for cancer immunotherapy.The human breast adenocarcinoma cell lines SK-BR-3 and MDA-MB-361 (American Type Culture Collection) were cultured in McCoy's 5a medium (Invitrogen) supplemented with 20% heat inactivated fetal calf serum , 100 units/ml penicillin (Invitrogen) and 100 μg/ml streptomycin (Invitrogen) and in Dulbecco's Modified Eagle's Medium  containing 20% FCS, 100 units/ml penicillin and 100 μg/ml streptomycin, respectively. Lenti-X 293T cells (Takara Bio Europe / Clontech) were maintained in DMEM supplemented with 10% FCS, 100 units/ml penicillin and 100 μg/ml streptomycin.6 cells/ml in RPMI 1640 Glutamax-I medium supplemented with 10% FCS, 100 units/ml penicillin and 100 μg/ml streptomycin.Experiments were approved by the Ethics Committee of the Christian-Albrechts-University , in accordance with the Declaration of Helsinki. Blood was drawn after receiving the donors’ informed consent. MNC were prepared as described previously . NK cellde novo according to published sequences (Eurofins MWG GmbH) [de novo according to published sequences [The variable regions derived from the humanized antibody humAb4D5-8 were employed for the generation of HER2-specific immunoconstructs , 47. TheWG GmbH) , 48. Theequences . The immequences . Concent2 (each from Abcam). PVR was detected with specific PE-coupled antibodies (R&D systems). Cell surface AICL was visualized with goat polyclonal anti-AICL antibodies and secondary FITC-labeled donkey anti-goat IgG (each from Santa Cruz Biotechnology). All antibodies were applied according to the manufacturer's recommendations and appropriate isotype-matched antibodies were used as controls. The fusion protein containing the ECD of NKp30 and the human IgG Fc domain (NKp30-Fc) was expressed and purified as described previously [Murine antibodies from hybridomas 4D5 , 225  and TH-111   were purYASARA Structure software (YASARA Biosciences) was employed to calculate the homology models for the HER2-specific scFv derived from antibody humAb4D5-8 and the ligands B7-H6, AICL and PVR individually. Secretion leader sequences and tags were removed. Structures for whole molecules were generated by introducing linker sequences and fusing the best-fitting models obtained for the single subunits. Ribbon drawings were generated using Discovery Studio 2.0 Visualize software (Accelrys Inc.).SDS-PAGE and Western transfer experiments were performed by standard procedures . The immImmunoligands were deglycosylated under denaturing reaction conditions using Protein Deglycosylation Mix (New England Bio Labs) containing the enzymes O-glycosidase, PNGase F, neuraminidase, β1–4 galactosidase and β-N-acetylglucosaminidase according to the manufacturer's instructions. For western blotting, 1.5 μg of proteins were loaded onto a gel.2 fragments (Beckman Coulter). Receptor expression levels were quantitated by determination of specific antigen binding capacities (SABC) employing Qifikit (DAKO) according to manufacturer's protocols.Flow cytometry was performed on a Navios flow cytometer (Beckman Coulter) as described earlier . For bin51Cr release assays performed in 96-well microtiter plates in a total volume of 200 μl as described previously [Cytotoxicity was analyzed in standard 3 h eviously . Human MP-values were calculated employing repeated measures ANOVA and the Bonferroni post-test, or the student's t-test when appropriate. P ≤ 0.05 were regarded as statistically significant. CI values were calculated with CalcuSyn software (Biosoft) according to the method of Chou and Talalay using the formula CIx = DA/DxA + DB/DxB [xA and DxB indicate doses of drug A and drug B alone producing x% effect, and DA and DB indicate doses of drugs A and B in combination producing the same effect.Graphical and statistical analyses were performed with GraphPad Prism 5.0 software. + DB/DxB , where D