Immunogenic Dendritic Cell Generation from Pluripotent Stem Cells by Ectopic Expression of Runx3

This information is current as Erika Takacs, Pal Boto, Emilia Simo, Tamas I. Csuth, of September 25, 2021. Bianka M. Toth, Hadas Raveh-Amit, Attila Pap, Elek G. Kovács, Julianna Kobolak, Szilvia Benkö, Andras Dinnyes and Istvan Szatmari J Immunol published online 16 November 2016

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Supplementary http://www.jimmunol.org/content/suppl/2016/11/15/jimmunol.160003 Material 4.DCSupplemental http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 16, 2016, doi:10.4049/jimmunol.1600034 The Journal of Immunology

Immunogenic Dendritic Cell Generation from Pluripotent Stem Cells by Ectopic Expression of Runx3

Erika Takacs,*,1 Pal Boto,*,1 Emilia Simo,* Tamas I. Csuth,* Bianka M. Toth,* Hadas Raveh-Amit,† Attila Pap,‡ Elek G. Kova´cs,x Julianna Kobolak,† Szilvia Benko¨,x Andras Dinnyes,†,{ and Istvan Szatmari*,‖

Application of dendritic cells (DCs) to prime responses to tumor Ags provides a promising approach to immunotherapy. However, only a limited number of DCs can be manufactured from adult precursors. In contrast, pluripotent embryonic stem (ES) cells represent an inexhaustible source for DC production, although it remains a major challenge to steer directional differentiation because ES cell–derived cells are typically immature with impaired functional capacity. Consistent with this notion, we found that mouse ES cell–derived DCs (ES-DCs) represented less mature cells compared with bone marrow–derived DCs. This finding prompted us to compare the expression profile of the ES cell– and adult progenitor-derived, GM-CSF–instructed, noncon- Downloaded from ventional DC subsets. We quantified the mRNA level of 17 DC-specific transcription factors and observed that 3 transcriptional regulators (Irf4, Spi-B, and Runx3) showed lower expression in ES-DCs than in bone marrow–derived DCs. In light of this altered , we probed the effects of these transcription factors in developing mouse ES-DCs with an isogenic expression screen. Our analysis revealed that forced expression of Irf4 repressed ES-DC development, whereas, in contrast, Runx3 improved the ES-DC maturation capacity. Moreover, LPS-treated and Runx3-activated ES-DCs exhibited enhanced T cell activation and migratory potential. In summary, we found that ex vivo–generated ES-DCs had a compromised maturation ability and immu- http://www.jimmunol.org/ nogenicity. However, ectopic expression of Runx3 enhances cytokine-driven ES-DC development and acts as an instructive tool for the generation of mature DCs with enhanced immunogenicity from pluripotent stem cells. The Journal of Immunology, 2017, 198: 000–000.

luripotent stem cells (PSCs), including embryonic stem differentiation capacity. PSC-derived functional cells can be (ES) cells and induced PSCs cells, provide an inexhaustible generated through directed differentiation using well-defined source for cell replacement and adoptive immune cell protocols (1). However, it is still challenging to steer the differ-

P by guest on September 25, 2021 therapy because of their unlimited self-renewal activity and broad entiation of PSCs to adult-like cells because the end products often represent embryonic-type or immature cells with limited activity. For example, human ES cell–derived RBCs readily *Stem Cell Differentiation Laboratory, Department of Biochemistry and Molecular expressed the embryonic and fetal globins (ε and g), but the adult Biology, Faculty of Medicine, University of Debrecen, H-4010 Debrecen, Hungary; †Biotalentum Ltd., H-2100 Go¨do¨llo,} Hungary; ‡Department of Biochemistry and b-globin was barely detected in these cells (2). Similarly, Molecular Biology, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, the gene expression signature of PSC-derived insulin-producing Hungary; xDepartment of Physiology, Faculty of Medicine, University of Debrecen, { cells mimicked that of fetal pancreatic tissue rather than the adult H-4010 Debrecen, Hungary; Molecular Animal Biotechnology Laboratory, Szent ‖ Istva´n University, H-2101 Go¨do¨llo,} Hungary; and Faculty of Pharmacy, University b cells (3). In addition, immaturity of the sarcoplasmic reticulum of Debrecen, H-4032 Debrecen, Hungary and diminished inotropic response to hormonal stimuli were de- 1E.T. and P.B. are cofirst authors. tected in murine ES cell– or induced PSC–derived cardiac cells ORCIDs: 0000-0002-8780-5199 (P.B.); 0000-0003-3300-8597 (H.R.-A.); 0000- (4). These findings suggest that embryonic developmental pro- 0001-7356-6345 (S.B.); 0000-0003-3791-2583 (A.D.). grams are readily activated in PSC-derived ex vivo–differentiated Received for publication January 6, 2016. Accepted for publication October 28, cells; however, these regulatory networks usually do not guarantee 2016. the production of fully active mature cells. For proper maturation, This work was supported by the University of Debrecen Faculty of Medicine Re- further steps are needed that are unknown or missing from the search Fund (Bridging Fund), Projects TA´ MOP-4.2.1/B-09/1/KONV-2010-0007 and TA´ MOP 4.2.2.A-11/1/KONV-2012–0023 (to I.S.), Project TA´ MOP-4.2.2/B-10/1- existing standard in vitro–differentiation protocols. 2010-0024 (to E.T.), EU FP7 Projects (EpiHealthNet, PITN-GA-2012-317146, IDP- Cell differentiation and commitment are governed by lineage- byNMR, PITN-GA-2010-264257), Research Center of Excellence Project 11476-3/ determining and stimulus-activated transcription factors (5). These 2016/FEKUT (to A.D.), and by Project OTKA K109429 (to S.B.). I.S. and S.B. were the recipients of a Bolyai Fellowship from the Hungarian Academy of Sciences. S.B. master regulators are gradually induced during the embryonic also was the recipient of a Ja´nos Szodoray Postdoctoral Fellowship from the Faculty development, and distinct factors modulate cell fate specification of Medicine, University of Debrecen. during the various stages of differentiation. In this study, we ex- Address correspondence and reprint requests to Dr. Istvan Szatmari, Department of amined the network in stem cell–derived dif- Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Egyetem ter 1, Debrecen, H-4010, Hungary. E-mail address: [email protected] ferentiated immune cells. GM-CSF–dependent dendritic cell (DC) The online version of this article contains supplemental material. development was selected as a differentiation model because this Abbreviations used in this article: BM, bone marrow; BM-DC, BM-derived DC; DC, cell type can be generated from adult stem cells and ES cells (6– dendritic cell; ES, embryonic stem; ES-DC, ES cell–derived DC; FSC, forward 13). Moreover, numerous transcription factors were described that scatter; MHCII, MHC class II; pDC, plasmacytoid dendritic cell; PSC, pluripotent control the commitment and specification of DCs (14, 15). In stem cell. addition, in vitro–generated DCs were applied in cell therapy– Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 based clinical trials to provoke anticancer immune responses (16).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600034 2 Runx3-INSTRUCTED DENDRITIC CELLS

Therefore, ex vivo DC manufacturing has an immediate biotech- T cell–proliferation analysis nological application. DC generation from PSCs is a promising Splenic T cells were isolated and purified from male BALB/c mice using a approach; however, PSC-derived DCs often exhibited a subopti- Pan T Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). mal T cell–activation capacity (6, 11, 13). Consistent with this The purified T cells were used as responders. For allogeneic MLRs, 103 or 4 5 notion, in this study we found that ES cell–derived DCs repre- 10 ES-DCs as stimulators were cocultured with 10 responders in wells of sented less mature cells compared with adult stem cell–derived 96-well round-bottom culture plates for 5 d. BrdU was added during the last 12 h of the culture. At the end of the culture, half of the cells were DCs. Furthermore, our gene expression analysis revealed that three centrifuged onto a 96-well plate, and the incorporation of BrdU was DC-affiliated transcription factors (IRF4, SPI-B, and RUNX3) were measured with a BrdU Cell Proliferation Assay Kit (Merck Millipore), poorly expressed in ES cell–derived DCs (ES-DCs). Remarkably, according to the manufacturer’s recommendations. improved DC maturation with enhanced chemotactic activity was Quantitative real-time RT-PCR detected on Runx3-instructed ES-DCs, suggesting that reintro- duction of a missing transcription factor can greatly enhance the RNA was isolated from ES cell– or BM-derived cells with TRI Reagent immunogenicity of PSC-derived APCs. (MRC), from which cDNA were reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Quantitative PCR was performed using a real-time PCR system (LightCycler 480; Roche), as Materials and Methods described previously (20). In brief, 40 PCR cycles were run at 95˚C for ES cell culture and cell differentiation 12 s and 60˚C for 30 s using TaqMan Gene Expression Assay primer-probe sets (Life Technologies). TaqMan assay IDs are shown in Supplemental Mouse ES cells were maintained on a mitomycin C (Merck Millipore, Table I. The comparative Cycle threshold method was used to quantify Darmstadt, Germany)–treated mouse embryonic fibroblast layer in knockout transcripts, and the expression level was normalized to b-actin. All PCR DMEM (Life Technologies, Carlsbad, CA) with 15% FBS qualified for ES reactions were done in triplicate with one control sample in which reverse Downloaded from cells (Biochrom, Cambridge, U.K.) and 1000 U/ml inhibitory transcriptase was omitted during cDNA synthesis. factor (Merck Millipore). ES-DCs were differentiated with a GM-CSF– dependent OP9 coculture method, as described (11) with minor modifi- Western blot analysis cations. In brief, OP9 cell density was set to 100,000 in a T25 flask 1 d Whole-cell extract of 15 mg of protein was separated by electrophoresis in before the experiment. Coculture was started by adding 100,000 harvested 10% polyacrylamide gel and then transferred to a polyvinylidene difluoride ES cells to the OP9 stromal cell layers; cells were cocultured for 5 d in membrane (Merck Millipore). Membranes were probed with anti-RUNX3 a http://www.jimmunol.org/ -MEM containing 20% FBS (Life Technologies). Half of the medium (GTX12343; GeneTex, Irvine, CA) or anti-IRF4 (sc-6059; Santa Cruz was replaced at day 3. At day 5, cells were harvested and reseeded onto Biotechnology, Dallas, TX) polyclonal Abs and then reprobed with anti- fresh OP9 layers. OP9 cell density was set to 40,000 cells per well in a six- GAPDH mAb (AM4300; Thermo Fisher Scientific, Waltham, MA). well plate 1 d before the experiment. A total of 200,000 5-d differentiated ES cells was added to the OP9 layers. Cells were cultured in a-MEM/20% ELISA FBS medium containing 50 ng/ml GM-CSF (PeproTech, Rocky Hill, NJ) and 50 mM 2-ME (Sigma, St. Louis, MO) for six additional days. At day Supernatants collected from ES-DCs or BM-DCs were evaluated for 11, floating and loosely adherent cells were harvested and further cultured production of cytokines (IL-1b, IL-6, and TNF-a) using DuoSet ELISA for 8 d in RPMI 1640 medium (Sigma) containing GM-CSF (50 ng/ml) kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s and 2-ME (50 mM) without OP9 cells. In the case of inducible ES cell– instructions. Detection limits for the kits are 15.6 pg/ml (IL-6 and IL-1b) derived cells (Irf4, Spi-B,orRunx3), 10-d differentiated CD45+ cells were and 31.25 pg/ml (TNF-a). For cytokine analysis, cell culture medium was sorted and further cultured for 9 d in RPMI 1640 medium containing replaced with fresh medium 24 h before harvesting the ES-DC or BM-DC by guest on September 25, 2021 GM-CSF (50 ng/ml) and 2-ME (50 mM). To induce DC maturation, supernatants. medium was replaced with fresh RPMI 1640 on day 18, and ES-DCs were treated with 100 ng/ml LPS. Endocytosis FITC–dextran 70 (Sigma; average molecular mass 70 kDa) was used to Bone marrow–derived cell isolation and differentiation measure mannose –mediated endocytosis. Immature ES-DCs or 3 5 Bone marrow (BM) cells were obtained from 12-wk-old male C57BL/6 or BM-DCs were incubated with 1 mg/ml FITC–dextran (1.5 10 DCs in 129S1 mice. The femurs and tibiae were removed, cleaned of all connective 24-well plate) for 1 h at 37˚C (control at 0˚C), and uptake was analyzed by tissue, and placed on ice in 2 ml of PBS. The ends of each femur and tibia flow cytometry. Phagocytosis was assessed by the cellular uptake of latex beads (Sigma; carboxylate modified, mean diameter 1 mm). Immature were clipped to expose the marrow. For BM-derived DC (BM-DC) dif- 3 5 ferentiation, 500,000 freshly isolated BM cells were cultured for 9 d in ES-DCs or BM-DCs were incubated with latex beads (1.5 10 DCs + 1.2 3 106 latex beads in a 24 well-plate) for 2 h at 37˚C (control at 0˚C), RPMI 1640 medium containing 10% FBS (Life Technologies), GM-CSF and uptake was quantified by flow cytometry. (50 ng/ml), and 2-ME (50 mM) in six-well tissue culture plates. Half of the medium was replaced every 3 d. To induce DC maturation, medium DC migration was replaced with fresh RPMI 1640 on day 8, and BM-DCs were treated with 100 ng/ml LPS. DC migration toward CCL19 and CCL21 was assessed using Transwell migration assays. Chemotaxis was tested using 24-well plates and inserts Construction of inducible cell lines with 5-mm pores (Corning-Sigma). A total of 600 ml of RPMI 1640 media with or without chemokines CCL21 (500 ng/ml; R&D Systems) and RNA was prepared from mouse splenic cells with TRI Reagent (MRC, CCL19 (500 ng/ml; R&D Systems) was placed in the lower chamber. Cincinnati, OH), and cDNAwas generated with a Transcriptor High Fidelity 5 Thereafter, 2 3 10 LPS-treated ES-DCs or BM-DCs were placed in the cDNA Synthesis Kit (Roche, Basel, Switzerland). The coding sequences of Transwell inserts (volume 100 ml) and allowed to migrate through a Spi-B Irf4 mouse and were amplified from splenic cell–derived cDNA polycarbonate mesh at 37˚C. After 4 h, cells that migrated to the lower using a FastStart High Fidelity PCR System (Roche), and the PCR prod- chamber were collected, and cell number was determined by acquiring Runx3 ucts were subcloned into the pDONR221 plasmid. The -carrying events for a fixed time period (3 min) using a constant flow rate with flow Gateway master vector (pENTR223.1; clone ID: FLH481113.01X) was cytometry. purchased from the DNASU plasmid depository. The obtained Gateway entry clones were recombined to a modified p2Lox plasmid (17, 18) Flow cytometry and cell sorting containing Gateway destination sequences using the Gateway Cloning System (Life Technologies). For ES cell transfection, 5 mg of the targeting Cells were analyzed and sorted with a FACSAria III (BD Biosciences, San constructs was electroporated into ZX1 (19) ES cells with the Neon Diego, CA). Live cells were gated based on a forward scatter (FSC)/side Transfection System (Life Technologies). Inducible cassette-exchange re- scatter profile to eliminate dead cells or cell debris. For FACS sorting, combination was used to insert the selected open reading frames into a ∼150,000 Flk1+ or 150,000–500,000 CD45+ cells were sorted. The con- euchromatic site on the X (17, 18). ES cell colonies were jugated Abs CD45-FITC (30-F11), CD11b-PE (M1/70), CD135-PE (A2 selected in 300 mg/ml G418 containing ES cell medium, picked on day 8, F10.1), CD11c-allophycocyanin (HL3), MHC2-FITC (I-A/I-E; 2G9), and expanded. At least five independent colonies per transgene were ex- CD80-allophycocyanin (16-10A1), and CD86-allophycocyanin (GL1) panded and characterized. were obtained from BD Biosciences. F4/80–Alexa Fluor 488 (BM8) Ab The Journal of Immunology 3 was purchased from eBioscience (San Diego, CA), and CD115-PE however, we consistently obtained lower expression of CD11c on (AFS98) was from BioLegend (San Diego, CA). Data analysis was ES-DCs. Next, we investigated the expression of CD135 and performed with BD FACSDiva 6.1.3 software (BD Biosciences). CD115, receptors for Flt3L and M-CSF, which are necessary for Statistical analysis the differentiation of conventional DCs and monocyte-derived All data are presented as mean 6 SD of the mean. Significant differences cells, respectively (23). Importantly, ES-DCs and BM-DCs con- + between mean values were evaluated using a two-tailed unpaired Student tained ,1% CD135 (FLT3) cells. In contrast, 40–60% of ES-DCs t test. and BM-DCs expressed CD115, a monocyte marker (Fig. 1B). These data suggest that ES- and BM-derived cells resemble the Results CD11b+ monocyte-derived inflammatory DC type (24); these ES-DCs exhibit a limited maturation capacity monocytic cells are ontogenetically unrelated to FLT3-dependent To accelerate our understanding of DC differentiation, we inves- conventional DCs. In addition, our flow cytometric analyses revealed tigated the efficiency of DC generation from mouse ES cells and that both cell types had a rather similar immune phenotype, although assessed the phenotype of the obtained products. There are a few ES-DCs tend to express less CD11c. protocols for directed DC differentiation from murine PSCs Next, we investigated the maturation/activation capacity of these (11–13, 21, 22). We applied a GM-CSF–driven OP9 coculture ex vivo–differentiated cells. Unexpectedly, maturation markers method that was established by Senju et al. (11). In brief, mouse (CD80 and MHC class II [MHCII]) were moderately induced upon ES cells were cocultured on OP9 stromal cells for 5 d; thereafter, LPS administration in ES-DCs: ,25% of the cells were double the obtained differentiated cells were transferred to new OP9 cell positive for MHCII/CD80. In contrast, .50% of the BM-DCs were layers and cultured for an additional 6 d in the presence of GM-CSF. double positive for MHCII/CD80 (Fig. 1C). In addition, we found Downloaded from At day 11, the loosely adhered and floating cells were transferred to that LPS-exposed ES-DCs exhibited a heterogeneous CD86 ex- new tissue culture flasks and cultivated for 8 d in the presence of pression; in contrast, the majority of LPS-activated BM-DCs were GM-CSF using feeder cell–free tissue culture conditions. To char- CD86+. These results suggest that ES-DCs represent a distinct acterize these ex vivo–generated immune cells, we compared the subset of myeloid cells that possess an impaired maturation ca- cell surface expression of ES-DCs with BM-DCs. Fig. 1A showed pacity. It is worth mentioning that in our study ES-DCs were de-

that CD45 and CD11b were readily detected on both cell types; rived from the E14 ES cell line (genetic background 129/Ola); in http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. ES-DCs possess a limited maturation capacity. Analysis of the 19-d cultured ES-DC (E14 ES cells were used) and 9-d cultured BM-DC (BM cells were obtained from C57BL/6 mice) phenotype. Flow cytometric profiles representative of at least three independent experiments are shown. The percentage of cells falling within the indicated quadrant or gate is shown. (A) Flow cytometric plots showing the expression of CD45, CD11c, and CD11b on immature DCs. (B) Cell surface expression of CD135 and CD115 on immature DCs. FSC is plotted on the y-axis. (C) Cell surface markers (MHCII, CD80, CD86, and F4/80) were assessed on 19-d differentiated ES-DCs and BM-DCs. The indicated cells were treated with 100 ng/ml LPS for 24 h before harvesting. 4 Runx3-INSTRUCTED DENDRITIC CELLS contrast, BM cells were isolated from C57BL/6 animals. To prove among the tested . In contrast, seven transcription factors that the altered maturation capacity and CD11c expression are in- were barely detected in ES-DCs (Batf3, Bcl6, Irf4, Mafb, Relb, dependent of the genetic background, ES-DCs derived from the Spi-B, and Zbtb46). Interestingly, those factors that were firmly B6 IA2 ES cell line (genetic background C57BL/6) were also in- detected at day 19 usually exhibited detectable transcript levels at vestigated. Again, impaired expression of CD11c/MHCII and CD86 day 11, implying that the expression of these DC-specific tran- was detected (Supplemental Fig. 1), demonstrating that ES-DCs scription factors was established during the early stage of myeloid tend to express less CD11c and represent less mature cells. To development. Altogether, these RNA profiling data indicated that further characterize these ex vivo–generated DC subsets, we in- several classical DC/-specific transcription factors vestigated the transcription factor constitution of these cells. were expressed in ES-DCs; however, other DC-specific genes were barely detected in these cells. It is important to note that our Gene expression signature of ES-DCs and their progenitors transcription factor selection included conventional and plasma- Tissue-specific transcription factors are important regulators of cytoid DC (pDC)-specific genes (e.g., Batf3, Spi-B, and Tcf4). cellular function; in addition, some of them have a direct role Therefore, the low transcript level of these factors was predictable in lineage commitment (25). To characterize the ES-DC–specific and expected in this FLT3-independent GM-CSF–driven DC- transcription factor profile, we quantified the RNA expression of differentiation model. 17 DC/macrophage-specific transcription factors (Batf3, Bcl-6, Egr1, Egr2, Id2, Ikzf1, Irf2, Irf4, Irf8, Maf, Mafb, Relb, Runx3, Irf4, Spi-B, and Runx3 are underexpressed in ES-DCs Pu.1/Sfpi, Tcf4, Spi-B, and Zbtb46) in ES-DCs and their progen- To further define the gene expression signature, mRNA levels of the itors. These selected genes had well-established roles in regulating selected transcription factors in ES-DCs were directly compared Downloaded from DC or macrophage development (14, 15). with BM-DCs. Interestingly, among the highly expressed genes Expression data were extracted from the starting pluripotent ES only Maf showed altered expression in ES-DCs; the transcript cells and the 5-, 11-, and 19-d differentiated cells. Of note, upon levels of the other eight genes were similar in both cell types differentiation, the emerging cells at days 5 and 11 represent a (Fig. 2A). These results suggests that a similar set of transcription mixed population; therefore, the putative DC progenitors were factors participated in the differentiation and maintenance of GM-

purified by cell sorting (Supplemental Fig. 2A). Our quantitative CSF–dependent myeloid DCs, regardless of cell origin. http://www.jimmunol.org/ mRNA transcript analysis indicated that more than half of the In contrast to the abundantly expressed transcripts, overt alter- investigated transcription factors (Egr1, Egr2, Id2, Ikzf1, Irf2, Irf8, ations were encountered when mRNA levels of the moderately Maf, Pu.1/Sfpi1, and Tcf4) exhibited a relatively high expression expressed genes were examined. Five genes exhibited lower ex- in the 19-d differentiated ES-DCs (Supplemental Fig. 2B). Re- pression in ES-DCs than in BM-derived cells (Fig. 2B). Of note, markably, Id2, Irf8, and Pu.1 were the most abundant transcripts the E14 mouse cell line (genetic background 129/OLA) was used by guest on September 25, 2021

FIGURE 2. Transcription factor expression profile of ES-DCs and BM-DCs. (A) Transcript profile of the highly expressed genes in ES-DCs compared with BM-DCs. Nineteen-day ES-DCs were obtained from E14 cells (genetic background: 129/OLA), and 9-d BM-DCs were derived from C57BL/6 animals. Transcript levels were determined with real-time quantitative PCR. The average gene expression levels and SD values were calculated from five independent experiments. (B) Transcript levels of low-expressing transcription factors in ES-DCs (E14) were compared with BM-DCs (C57BL/6). (C) Gene expression data were derived from additional independent cell sources. ES-DCs were generated from R1 ES cells (genetic background: 129X1 3 129S1), and BM-DCs were derived from 129/S1 mice. The average gene expression levels and SD values were calculated from four or five independent exper- iments. (D) IRF4 protein detection with immunoblot testing two ES-DC and two BM-DC protein extracts. The identity of specific bands was confirmed by comigration with a band seen in the extract of Irf4-induced cells (Irf4 +dox). GAPDH was used as a loading control. (E) RUNX3 protein detection with immunoblot testing an ES-DC and BM-DC protein extract. The identity of specific bands was confirmed by comigration with a band seen in the extract of Runx3-induced cells (Runx3 +dox). *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 5 for ES-DC generation, whereas BM-DCs were derived from were tested in all cases. Unexpectedly, much fewer CD45/CD11b C57BL/6 animals. To prove that the observed alterations are in- double-positive cells were detected upon the forced expression of dependent of the genetic background, DCs were differentiated Irf4 (Fig. 3A), suggesting that this transcription factor exerted a from additional stem cell sources. ES-DCs were derived from R1 detrimental effect on the early stage of ES-DC development. In (genetic background 129X1/SvJ 3 129S1) ES cells, and BM-DCs contrast, more CD45/CD11b+ cells were differentiated upon ac- were obtained from 129S1 animals. Importantly, four genes tivation of Spi-B or Runx3 (Fig. 3B, 3C). These results imply that showed significantly altered expression in this independent sample ectopic expression of Spi-B or Runx3 enhances the GM-CSF– set: Spi-B, Irf4, and Runx3 had lower expression in ES-DCs than driven myeloid differentiation in ES cell–derived progenitors. in BM-DCs, whereas higher Maf transcript levels were detected in Forced expression of Runx3 is coupled with enhanced DC ES-DCs (Fig. 2C). maturation Next, we evaluated the protein expression of Irf4 and Runx3, whose RNA transcripts exhibited a relatively high expression in We next examined the effects of these three factors on fully dif- BM-DCs. In agreement with the RNA profiles, we observed de- ferentiated ES-DCs. In this set of transgenic experiments, tran- tectable levels of IRF4 and RUNX3 from BM-DCs sam- scription factors were turned on between days 5 and 19. In addition, ples, but these proteins were barely detected in ES-DCs (Fig. 2D, cells were treated with LPS at day 18 to stimulate DC maturation/ 2E). In conclusion, most of the tested myeloid/DC-specific tran- activation. It is important to mention that, in contrast to the previous + scription factors exhibited similar expression patterns in ES- and experiments (Figs. 1, 2), CD45 cells were sorted at day 10, and BM-derived DCs. However, three transcription factors were these purified myeloid cells were differentiated further until day underexpressed in ES-DCs, suggesting that upregulation of these 19. We found that this sorting step was necessary to recover the Downloaded from factors might modify the differentiation and function of these Runx3-expressing hematopoietic progenitors because these cells immune cells. tend to adhere firmly to the OP9 layers at days 10–11. First, we tested the impact of Irf4 on the 19-d differentiated ES-DCs. Notably, Spi-B– and Runx3-dependent enhanced myeloid development only a few live cells were detected from 200,000 CD45+ sorted cells In light of this altered gene expression, we probed the effects of the in the presence of doxycycline (data not shown). The poor yield

underexpressed transcription factors (Spi-B, Irf4, and Runx3)in precluded a detailed phenotypic analysis, and this result indicates that http://www.jimmunol.org/ developing ES-DCs using isogenic expression screening. We ap- Irf4 exerts a general inhibitory effect on ES-DC development. plied a genetically modified mouse ES cell line (ZX1), into which We then assessed the phenotype of the Spi-B–andRunx3- a gene of interest can be inserted by Cre-mediated recombination programmed APCs. To evaluate the maturation of the final differ- (17–19). In these cells, the investigated genes can be induced in entiation products, cell surface expression of CD80 and MHCII response to tetracycline or doxycycline (Tet-on system). This in- were determined in the presence or absence of LPS. As expected, ducible system was used previously to modulate ES cell–derived augmented CD80/MHCII+ cell formation was obtained upon LPS blood, skeletal muscle, or thyroid development via induction of stimulation. However, overexpression of Spi-B failed to modify lineage-specific transcription factors (26–28). the maturation capacity of ES-DCs, because the frequency of + To evaluate the early effects of Irf4, Spi-B,orRunx3 on myeloid MHCII/CD80 cells was unaltered in the presence of doxycycline by guest on September 25, 2021 blood cell development, the inducible ES cell clones were dif- (Supplemental Fig. 3). In striking contrast, an elevated percentage ferentiated for 11 d with the OP9 coculture method. The selected of MHCII/CD80+ cells was detected in Runx3-instructed DCs upon transgenes were turned on during the second stage of differenti- LPS treatment (Fig. 4). Moreover, we observed a distinct MHCII- ation (days 5–11). Two independent ES cell clones per transgene expressing subpopulation of ES-DCs in the presence of RUNX3,

FIGURE 3. Spi-B– and Runx3- dependent enhanced myeloid develop- ment. Inducible ES cells were cultured for 11 d, as described in Materials and Methods. To turn on the indicated transgenes, cells were treated at day 5 with 1 mg/ml doxycycline (+ dox). The differentiated cells were harvested and analyzed at day 11. Cell surface phenotype (CD45 and CD11b) of Irf4- activated (A), Spi-B–activated (B), and Runx3-activated (C) cells were assessed. Flow cytometric profiles representative of three independent experiments are shown. The percentage of cells falling within the indicated quadrant is shown. Two ES cell clones per transgene were tested (Irf4: C1 and C3; Spi-B: C2 and C4; Runx3: C2 and C4). 6 Runx3-INSTRUCTED DENDRITIC CELLS Downloaded from http://www.jimmunol.org/

FIGURE 4. Runx3-driven enhanced MHCII expression. Runx3-transgenic ES cells were differentiated for 19 d, as described in Materials and Methods. (A) Runx3-inducible cells were treated with 1 mg/ml doxycycline (+ dox). In addition, the indicated cells were treated with LPS (100 ng/ml) at day 18. Cell surface expression of CD80 and MHCII was assessed by flow cytometry at day 19. Representative flow cytometric data were obtained from two Runx3- transgenic cell clones (C2 and C4). (B) Runx3-inducible cells (ES-DC Runx3) were differentiated as in (A). In addition, 9-d differentiated BM-DCs were analyzed. The indicated BM-DCs were treated with LPS (100 ng/ml) at day 8. Cell surface expression of CD80 and MHCII was assessed by flow cytometry.

The average percentage of CD80/MHCII double-positive cells and SD values were calculated from eight (ES-DC) or six (BM-DC) independent experi- by guest on September 25, 2021 ments. *p , 0.05, ***p , 0.001. even without LPS administration (Fig. 4A). These results sug- compared with control ES-DCs (Fig. 6A). These results suggest gested that the sustained expression of Runx3 potentiates the that immature ES-DCs had a profound phagocytic- and receptor- maturation capacity of ES-DCs; however, this MHCII/CD80 ex- mediated endocytic activity that was not modified or negatively pression level was still lower compared with BM-DCs (Fig. 4B). regulated, respectively, by Runx3. To further address the maturation ability of LPS-dependent DCs, We next tested the cytokine-production capacity of ES-DCs we examined the expression of CD86, a well-established DC using ELISAs. Remarkably, LPS-treated ES-DCs released high maturation marker, on Runx3-instructed ES-DCs. In line with amounts of IL-6 and TNF-a; in addition, IL-1b was readily de- MHCII/CD80 upregulation, more CD86+ cells were obtained in tectable in these mature cells (Fig. 6B). Of note, a similar cytokine LPS-treated Runx3-activated ES-DCs (Fig. 5). This finding con- production potential was obtained with or without doxycycline firmed that Runx3 positively regulates the maturation/activation treatment (only a nonsignificant reduction in IL-1b secretion was capacity of ex vivo–differentiated ES-DCs. observed in Runx3-activated ES-DCs). Furthermore, comparable cytokine levels were detected in supernatants derived from BM-DCs. Runx3 endows ES-DCs with enhanced migratory and T cell These findings indicate that our GM-CSF–driven inflammatory DC activation capacity subsets have a strong and universal ability to secrete proin- Finally, we examined the functional characteristics of Runx3- flammatory cytokines. In addition, we conclude that, in contrast to instructed ES-DCs. First, we assessed the endocytic activity of the well-established surface maturation markers, proinflammatory these transgenic cells by two methods: engulfment of latex beads cytokine production by ES-DCs was not altered by Runx3. for detection of phagocytosis and internalization of FITC-dextran, Next, we evaluated the CCL19/CCL21-dependent migratory which is mainly taken up by mannose receptor–mediated endo- ability of mature ES-DCs using Transwell-migration assays. After cytosis. Our flow cytometric analysis revealed that immature 4 h of incubation, transmigrated cells were enumerated by flow (non–LPS-treated) ES-DCs efficiently accumulated latex beads cytometry. Interestingly, ES-DCs migrated very poorly in the ab- after 2 h of incubation and that Runx3 induction did not influence sence of doxycycline treatment, even in the presence of chemokines the latex beads uptake capacity of these cells (Fig. 6A). Further- (CCL19 plus CCL21). In contrast, Runx3-activated ES-DCs trans- more, BM-DCs possessed a comparable phagocytic activity (latex migrated efficiently in the presence of chemokines (Fig. 7). To beads uptake capacity). In contrast, induction of Runx3 negatively compare the potency of ES- and BM-derived cells, we also assayed modulated the FITC-dextran uptake capacity of ES-DCs; fur- the migration potential of BM-DCs. Interestingly, these cells ex- thermore, BM-DCs also exhibited lower FITC-dextran uptake hibited a stronger migration, even in the absence of chemokines. The Journal of Immunology 7

FIGURE 5. Runx3-dependent en- hanced DC maturation. (A) Runx3 inducible cells were differentiated for 19 d, and the indicated samples were treated with 1 mg/ml doxycy- cline (+ dox). In addition, the indi- cated cells were treated with LPS (100 ng/ml) at day 18. Cell surface expression of CD86 and F4/80 was assessed by flow cytometry at day 19. Representative flow cytometric data were obtained from two Runx3- transgenic cell clones (C2 and C4). (B) Runx3-inducible cells (ES-DC Runx3) were generated as in (A). In addition, 9-d differentiated BM- DCs were analyzed. The indicated BM-DCs were treated with LPS (100 ng/ml) at day 8. The average Downloaded from percentage of CD86+ cells and SD values were calculated from eight (ES-DC) or six (BM-DC) indepen- dent experiments. *p , 0.05, ***p , 0.001. http://www.jimmunol.org/

We conclude that Runx3-activated ES-DCs possess an enhanced agreement with the elevated expression of MHCII (Fig. 4B), this migratory capacity although it is still inferior to that of their observation suggests that BM-DCs possess an even higher T cell– BM-derived counterparts. Finally, the T cell–activation capacity of activation capacity. ES-DCs was evaluated by MLRs using DCs as stimulators. Con- Collectively, these results indicate that Runx3-programmed sistent with the enhanced maturation capacity of Runx3-express- ES-DCs exhibited an elevated migratory capacity and possessed a ing cells, we observed an elevated T cell proliferation upon superior T cell–priming activity, although this potential is still lower by guest on September 25, 2021 doxycycline treatment. Of note, we also tried to test the T cell– compared with the abilities of BM-DCs. These functional data, priming capacity of BM-DC samples; however, some of the ab- together with the phenotypic profile, support the conclusion that sorbance values were out of the recommended linear range. In enforced expression of Runx3 improves ES-DC immunogenicity.

FIGURE 6. ES-DCs had prominent cytokine production and endocytic capacity. (A) The endocytic capacity of Runx3-inducible immature ES-DCs (ES-DC Runx3) and BM-DCs was evaluated by measuring the uptake of latex beads or FITC-dextran, as described in Materials and Methods. The indicated ES-DCs were treated with 1 mg/ml doxycycline (+dox). Mean fluorescence intensity (MFI) values were determined with flow cytometry. The average of MFI and SD values were calculated from three independent experiments. (B) Concentrations of the indicated cytokines (IL-6, TNF-a, and IL-1b) in the cultured supernatant were determined by ELISA. Runx3-inducible cells (ES-DC Runx3) and BM-DCs were generated as described in Materials and Methods. The indicated ES-DCs were treated with 1 mg/ml doxycycline (+dox). Supernatants were col- lected 24 h after the addition of LPS (100 ng/ml) or control medium. The average cytokine concentrations and SD val- ues were calculated from eight independent experiments. **p , 0.01, ***p , 0.001. 8 Runx3-INSTRUCTED DENDRITIC CELLS Downloaded from http://www.jimmunol.org/

FIGURE 7. Runx3-dependent enhanced DC migration and T cell activation. (A) The migratory capacity of Runx3-inducible mature (LPS-treated) ES-DCs and BM-DCs was evaluated by enumerating the cells in the lower chamber using Transwell migration wells. Cell migration was assayed in the by guest on September 25, 2021 presence or absence of chemokines (CCL19 and CCL21). The indicated ES-DCs were treated with 1 mg/ml doxycycline (+dox). Cells were enumerated with a flow cytometer, and representative flow cytometric data are presented. Numbers correspond to the gated events in 3 min. FSC is plotted on the x-axis, and side scatter (SSC) is plotted on the y-axis. (B) The average number of migrated cells and SD values were calculated from six (ES-DC) or three (BM-DC) independent experiments. Cell migration was assessed in the presence (+CC) of absence (2CC) of chemokines (CCL19 and CCL21). (C) T cell– proliferation capacity was tested by MLR. The indicated number of 19-d differentiated Runx3-inducible mature ES-DCs was used as stimulators for MLR. LPS-activated ES-DCs were cocultured with 105 splenic T cells isolated from BALB/c mice for 5 d, and the indicated samples were treated with 1 mg/ml doxycycline (+Dox). The proliferative response of T cells was assessed with a BrdU Cell Proliferation Assay. The corrected absorbance (450–540 nm) and SD values were calculated from four independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

Discussion had an impaired maturation ability compared with BM-derived Novel generation of DC vaccines must build on the increased cells. Importantly, we tested several ES cell lines, and we always knowledge of cell manipulation, including the efficient production observed an impaired maturation capacity (without transgene in- of various DC subsets ex vivo. GM-CSF–instructed BM-DCs, which duction). It is possible that minor differences in the protocols have were used in this study as control cells, are considered monocytic an impact on the final cell products. Moreover, in earlier studies (6, inflammatory DCs (29). Consistent with previous findings (11, 12, 11, 13), ES-DCs often exhibited a lower T cell–activation potential 21), we confirmed that ES-DCs resembled this nonconventional compared with the adult counterparts, suggesting that impaired inflammatory DC subset, because the final differentiation products immunity is a general characteristic of PSC-derived DCs. were CD11b+, and they efficiently produced proinflammatory cy- The prominently distinct phenotype of ES-DC prompted us to tokines upon LPS treatment. However, we also observed apparent examine the gene expression signature of DC-specific transcription differences between ES-DCs and BM-DCs. For example, lower factors in both ES- and BM-DCs. A handful of DC/macrophage- expression of CD11c was detected in ES-DCs; in addition, ES-DCs affiliated transcription factors was recently identified and charac- exhibited impaired expression of several maturation-specific terized (14, 15, 30); however, the contribution of these master markers upon stimulation with LPS. It is worth mentioning that regulators to the development of PSC-derived DCs has not been a similar phenotype was obtained whether DCs were differentiated investigated. Our focused analysis revealed that the expression of from 129/Ola (E14) or C57BL/6 (B6 IA2) animal-derived ES three DC-affiliated transcription factors (Spi-B, Runx3, and Irf4) cells, suggesting that these alterations are independent of the ge- was impaired in mouse ES-DCs compared with BM-DCs. These netic background of the starting mouse pluripotent cells. factors were already implicated in DC development. Irf4 is a Of note, most previous studies suggested that ES-DCs are prominent transcriptional regulator of CD11b+ conventional DCs functionally equivalent to adult stem cell–derived DCs. In contrast, (31–34). In addition, GM-CSF–driven BM-DC differentiation is we observed that ES-DCs exhibited a distinct phenotype, and they Irf4 dependent (35). Unexpectedly, our analysis failed to support The Journal of Immunology 9 the central role of this factor in ES cell–derived cells, because the a similar gene-induction strategy will be applicable to human cells, baseline expression of this gene was rather low in ES-DCs. In and these results pave the way for the future clinical application of addition, we observed impaired myeloid blood cell and DC de- PSC-derived DCs for adoptive immune cell therapy. velopment upon the forced expression of this transgene in ES-DC progenitors. It is worth mentioning that Irf8 is an abundant tran- Acknowledgments script in ES-DCs; therefore, our observations raised the possibility We thank Iren Mezo for excellent technical help and Michael Kyba (Uni- that Irf4 induction might interfere with the putative Irf8-driven versity of Minnesota, Minneapolis, MN) for providing the OP9 cells, the ES-DC developmental program. mouse ES cell lines (ZX1, E14, and R1), and the p2Lox plasmid construct. In contrast to Irf4, Spi-B exerted a positive effect on myeloid blood cell development. Enhanced production of CD45/CD11b+ cells was obtained after the upregulation of this transcription Disclosures factor during the early stage of differentiation. Interestingly, it was The authors have no financial conflicts of interest. reported that Spi-B is highly expressed in pDCs, and gene- silencing studies revealed that this factor was necessary for hu- References man pDC development (36, 37). In this study, we failed to obtain 1. Murry, C. E., and G. 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