A method for the genetic modification of dendritic cells (DC) was previously established based on the in vitro differentiation of embryonic stem (ES) cells to DC (ES-DC). The unavailability of human ES cells genetically identical to the patients will be a problem in the future clinical application of this technology. This study attempted to establish a strategy to overcome this issue. The TAP1 or β2-microglobulin (β2m) gene was disrupted in 129 (H-2b)-derived ES cells and then expression vectors for the H-2Kd or β2m-linked form of Kd (β2m-Kd) were introduced, thus resulting in two types of genetically engineered ES-DC, TAP1−/−/Kd ES-DC and β2m−/−2m-Kd ES-DC. As intended, both of the transfectant ES-DC expressed Kd but not the intrinsic H-2b haplotype-derived MHC class I. β2m−/−2m-Kd and TAP1−/−/Kd ES-DC were not recognized by pre-activated H-2b-reactive CTL and did not prime H-2b reactive CTL in vitro or in vivo. β2m−/−2m-Kd ES-DC and TAP1−/−/Kd ES-DC had a survival advantage in comparison to β2m+/−2m-Kd ES-DC and TAP1+/+/Kd ES-DC, when transferred into BALB/c mice. Kd-restricted RSV-M2-derived peptide-loaded ES-DC could prime the epitope-specific CTL upon injection into the BALB/c mice, irrespective of the cell surface expression of intrinsic H-2b haplotype-encoded MHC class I. β2m−/−2m-Kd ES-DC were significantly more efficient in eliciting immunity against RSV M2 protein-expressing tumor cells than β2m+/−2m-Kd ES-DC. The modification of the β2m or TAP gene may therefore be an effective strategy to resolve the problem of HLA class I allele mismatch between human ES or induced pluripotent stem cells and the recipients to be treated.

An efficient means for the activation of the CTL reactive to tumor Ags is crucial for T cell-mediated antitumor immunotherapy (1). Dendritic cells (DC)3 are potent T cell stimulators and cellular vaccination using Ag-loaded DC has proven to be an efficient means for priming CTL specific to Ags (2, 3). This laboratory and others have established methods to generate DC from mouse and human embryonic stem (ES) cells (4, 5, 6, 7, 8). The capacity of ES cell-derived DC (ES-DC or esDC) to simulate alloreactive T cells and to prime Ag-specific CTL is comparable to that of conventional bone marrow-derived DC (BM-DC). Genetically modified ES-DC can be readily generated by introducing expression vectors into ES cells and the subsequent induction of their differentiation into ES-DC (9). The transfection of ES cells can be done by electroporation with plasmid vectors and the use of virus-based vectors is not necessary. Once an ES cell clone with proper genetic modification is established, it then serves as an infinite source for genetically modified DC. Mouse models have demonstrated that vaccination with genetically engineered ES-DC expressing tumor Ags (10) and T cell-attracting chemokines (11) is very effective for the induction of antitumor immunity.

In the future, the clinical application of ES-DC technology will require a solution to the problem of histoincompatibility between patients to be treated and the ES-DC. Specifically, the HLA allele mismatch may cause a rapid immune response and rejection of the inoculated cells (12), although a discrepancy in the minor histocompatibility Ag can also be a cause of alloreaction (13). The present study addressed this problem by using the strategy of modification of the genes that control the cell surface expression of MHC class I, i.e., β2-microglobulin (β2m) and TAP. TAP1−/− and β2m−/− ES cell clones were generated from the ES cell lines derived from the 129 mouse (H-2b) embryo. The expression vectors for H-2Kd and β2m-linked form of H-2Kd (β2m-Kd) were then introduced into TAP1−/− and β2m−/− ES cell clones, respectively. Subsequently, these genetically modified ES cells were subjected to a differentiation culture to generate ES-DC. The MHC class I molecules encoded by the genes in the H-2b haplotype were either absent or at very low levels on the cell surface of these genetically modified ES-DC. The effect of the alteration of cell surface expression of MHC class I on activation of alloreactive (H-2b haplotype-encoded MHC class I-reactive) CTL was analyzed in both in vitro and in vivo experiments. After loading ES-DC with the antigenic peptide having a capacity to bind H-2Kd molecule, these ES-DC were transferred into BALB/c mice (H-2d haplotype) to determine whether the peptide-specific, H-2Kd-restricted CTL could be primed in the recipient mice and whether Ag-specific antitumor immunity could be induced.

Six- to eight-week-old female BALB/c and 129/Sv (129) mice were purchased from Japan SLC and Clea Japan, respectively. The mice were housed at the Center for Animal Resources and Development (CARD, Kumamoto University) under specific pathogen-free conditions. All studies were performed under the approval of the animal experiment committee of Kumamoto University.

The respiratory syncytial virus (RSV) M282–90 epitope (SYIGSINNI) restricted to H-2Kd has been described previously (14). H-2Kd-restricted HIV gag protein-derived p24199–207 epitope (AMQMLKETI) was used as an irrelevant control peptide (15). The peptides were commercially synthesized and supplied at >98% purity (Anygen). Murine mastcytoma P815 cells were used as target cells for a 51Cr release assay. A RSV-M2-transduced colon26/luciferase (Luc) cell line (colon26/M2-Luc) was established by the transfection of murine adenocarcinoma colon26 cells with an influenza virus hemagglutinin (HA)-tagged RSV M2 expression vector (pCAG-M2- internal ribosomal entry site (IRES)-neoR) and a firefly luciferase expression vector (pCAG-luc-IRES-puroR) by electroporation. After the transfection, G418 and puromycin were added to the culture medium for selection and single clones were obtained by limiting dilution. The expression of firefly luciferase was verified by measuring the luciferase activity in the cell lysates as described below. The expression of the HA-RSV-M2 protein in the selected transfectant clones was confirmed by a flow cytometric analysis following intracellular anti-HA staining and also using ELISA detecting IFN-γ-production by M282–90 specific Kd-restricted CTL cocultured with the transfectants.

ES cells were cultured on primary mouse embryonic fibroblast (PEF) feeder layers in complete ES cell medium, DMEM containing 20% KnockOut Serum Replacement (Invitrogen Life Technologies), 2-ME (50 μM), and mouse leukemia inhibitory factor (1000 U/ml). Α β2m+/− ES cell clone established from D3 cell line derived from a 129 mouse embryo (H-2b) was a generous gift from Dr. R. Jaenisch (Massachusetts Institute of Technology, Cambridge, MA) (16). To generate β2m−/− ES cells, β2m+/− ES cells (5 × 105 cells/90-mm culture dish) were cultured on feeder layers of neomycin-resistant PEF derived from GTPBP1−/− mouse embryos (17) in ES cell medium containing high dose G418 (1.5–2.0 mg/ml) for 10 days (18). After a further culture for 7 days without G418, the surviving ES cell colonies were picked up from the dishes, transferred to 24-well culture plates, and then expanded. For each isolated ES cell clones, a part of the expanded cells were cultured in gelatin-coated 6-well plates without PEF feeders. Genomic DNA extracted from the feeder-free ES cells was used for genotyping of the β2m locus by genomic PCR and β2m−/− ES clones were selected. TAP1+/− ES cells were generated from E14 ES cells derived from 129 mouse embryo (H-2b). E14 cells were transfected with 30 μg of linearized targeting vector by electroporation (19). G418 and ganciclovir were added to the culture medium 24 h after the transfection and the surviving colonies were isolated during days 7–10 of selection. The isolated clones were analyzed by PCR and Southern blotting to identify cell clones with homologous recombination. Subsequently, one of the TAP1+/− clones was subjected to selection with high dose of G418 as in the case of β2m−/− ES cells. The clones were expanded and analyzed by Southern blotting to select TAP1−/− clones. Expression vectors for H-2Kd and β2m-linked form of H-2Kd (β2m-Kd) were introduced into TAP1−/− and β2m−/− ES cell clones, respectively. The induction of differentiation of ES cells into ES-DC was done as described previously (20). On day 17–19 of cultures, the floating or loosely adherent cells were recovered from culture dishes by pipeting and then were used for the experiments.

The initial screening of ES cells was done by PCR using the following primers: the wild type TAP1 allele (5′-ATGGGACACATGCACGGC-3′ and 5′-CCACAGTAGCAGGCTCAG-3′), the mutant TAP1 allele (5′-TGTAGCTTTGGCTCTTCTGGAA-3′ and 5′-GGGCCAGCTCATTCCTCCACTC-3′), β2m (5′-CCTCAGAAACCCCTCAAATTCAAG-3′ and 5′-GCTTACCCCAGTAGACGGTCTTGG-3′). The set of primers for β2m was designed to amplify both the wild type and targeted loci.

To analyze the genotype of ES cells, genomic DNA isolated from ES cells was digested with XhoI and EcoR V. The DNA fragments were separated by electrophoresis in 0.8% agarose gels. Subsequently, the DNA was transferred onto nylon membranes. Probes for the Southern blot analysis were obtained by PCR with sets of primers for TAP1 locus (5′-GACCAGACTCTGGACAGCTCAC-3′ and 5′-AAGGCAAGAGAGAATCAAGAG-3′) from the genomic DNA of ES cells. Labeling of probe DNA with 32P-dCTP was done by using a Megaprime DNA Labeling Kit (GE Healthcare) and the standard Southern blot procedure was conducted.

FITC-conjugated anti-H-2Kd (BD Pharmingen), H-2Kb (Caltag Laboratories), and H-2Db (Caltag Laboratories) Abs were purchased from the indicated sources. A flow cytometric analysis was done on a FACScan flow cytometer (BD Biosciences) and the data analysis was performed using the CellQuest software program (BD Biosciences).

Bone marrow cells prepared from BALB/c or 129 mice were cultured in RPMI 1640 medium supplemented with 10% FCS, 500 U/ml GM-CSF, and 50 μM 2-ME. On day 7 of the culture, the cells were recovered and used as BM-DC for the experiments.

To generate BALB/c (H-2d)-derived H-2b-reactive CD8+ T cell lines, 5 × 106 BALB/c spleen cells were cultured with 2 × 106 irradiated 129 (H-2b) spleen cells in 2 ml of RPMI 1640 medium supplemented with 10% FCS, 100 U/ml IL-2 and 50 μM 2-ME in a well of 24-well plates for 5 days (21) and after that CD8+ T cells were isolated by using anti-CD8 magnetic beads (Miltenyi Biotec). The H-2b-reactive CD8+ T cells (1 × 103) were cultured with the indicated stimulator DC (5 × 103) for 16 h and the activation of T cells was detected by IFN-γ-production by using ELISPOT (BD Biosciences). For the analysis of the priming of alloreactive (H-2b-reactive) CD8+ T cells by ES-DC, BALB/c spleen cells (5 × 106) were cultured with irradiated ES-DC (2 × 106) for 5 days and then CD8+ T cells were isolated as described above. The magnitude of priming of H-2b-reactive CD8+ T cells was analyzed by IFN-γ-production detected by ELISPOT upon coculture with 129 mice-derived BM-DC as stimulators.

Spleen cells were isolated from naive BALB/c mice or those that received multiple ES-DC injections and CD8+ T cells were isolated by using anti-CD8 magnetic beads (Miltenyi Biotech). To analyze the frequency of auto (H-2d)- or allo (H-2b)-reactive CD8+ T cells, the cells (5 × 103) were cocultured with BALB/c or 129 BM-DC (5 × 103) for 16 h and IFN-γ producing cells were detected using the ELISPOT assay.

The in vivo elimination of ES-DC was assessed according to the reported procedures with some modification (22). In brief, ES-DC were labeled with the 10 μM chloromethyl-benzoyl-amino-tetramethyl-rhodamine (CMTMR; Molecular Probes) and BALB/c BM-DC were labeled with 10 μM chlorometylfluorescein diacetate (CMFDA; Molecular Probes) according to the manufacturer’s instructions. The mice were injected s.c. in the forelimb with 2 × 106 cells containing equal numbers of CMTMR-labeled ES-DC and CMFDA-labeled BM-DC. After 48 h, the draining axillary and brachial lymph nodes were removed, digested with collagenase type II and DNase I and analyzed for the presence of fluorescent cells by flow cytometry. The number of ES-DC was normalized to control syngeneic BALB/c BM-DC.

ES-DC were incubated with RSV-M282–90 peptide (10 μM) for 3 h and then washed three times with FCS-free DMEM. Ag-loaded ES-DC were injected i.p. (1 × 105 cells/injection/mouse) into the mice twice, with a 7-day interval. In some experiments, non-Ag-loaded ES-DC were injected five or ten times with 7-day intervals before the injection of Ag-loaded ES-DC. Seven days after the last injection of ES-DC, the mice were sacrificed and the spleen cells were isolated. After hemolysis, the spleen cells were cultured in the presence of M282–90 peptide (1 μM). Six days later, the cells were recovered and cytotoxic activity against M282–90 peptide pulsed-P815 target cells were measured using the standard 51Cr- release assay.

The mice were immunized with ES-DC and, 7 days after the immunization, colon26/M2-Luc (1 × 106/mouse) cells were injected into the mice i.p. Ten days later, the mice were sacrificed and the luciferase activity of the lysates of the abdominal organs was measured to quantify tumor growth. The tissue specimens were homogenized in 3 ml of lysis buffer (0.05% Triton X-100, 2 mM EDTA, 0.1 M Tris (pH 7.8)) and the homogenates were cleared by centrifugation at 10,000 × g for 5 min. Fifty μl of the supernatant was mixed with 50 μl of dilution buffer (PBS containing 2.4 mM CaCl2 and 0.82 mM MgSO4) and 100 μl of luciferase assay buffer (Steadyliteplus, PerkinElmer) and at 5 min after the mixing the light produced was measured for 1 second in a luminometer (Tristar LB941, Berthold Technologies).

Student’s t test was used for the statistical analysis of data except for the data regarding the tumor invasion experiments. Because some of data in the tumor invasion experiments did not follow a normal distribution, the data were analyzed using the Mann-Whitney U test, a nonparametric test. A value of p < 0.05 was considered to be significant.

The present study evaluated a strategy to prime Ag-specific CTL by transfer of genetically engineered 129 (H-2b)-derived ES-DC into BALB/c (H-2d) recipient mice, thus avoiding the recognition of ES-DC by allo (H-2b)-reactive CD8+ T cells. To modify the cell surface expression of MHC class I, two strategies were tested in parallel: 1) disruption of the β2m gene in ES cells and introduction of β2m-linked form of recipient-matched MHC class I (β2m-Kd) (Fig. 1,A), 2) disruption of TAP1 gene, introduction of recipient-matched MHC class I (Kd) and loading of Kd-binding epitopes to the ES-DC (Fig. 2 A).

FIGURE 1.

Generation of ES-DC deficient in the intrinsic β2m gene and expressing recipient-matched MHC class I linked to β2m. A, Overview of the method for generation of ES-DC-expressing β2m-linked Kd, β2m+/−2m-Kd ES-DC and β2m−/−2m-Kd ES-DC. β2m−/− ES cells were generated from β2m+/− ES cells by high dose G418 selection. β2m+/− and β2m−/− ES cells were introduced with cDNA for the β2m-linked form of Kd and subsequently differentiated to generate β2m+/−/β2m-Kd ES-DC and β2m−/−/β2m-Kd ES-DC. B, The absence of wild-type β2m gene in the β2m−/− ES cell clone was confirmed by genomic PCR. C, Structure of the expression vector for β2m-linked H-2Kd (left) and a schematic representation of the encoded molecule (right). The β2m was fused to H-2Kd via a flexible 15 amino acid-long linker ([Gly4-Ser]3). The vector is driven by CAG promoter (pCAG) and cDNA for β2m-linked H-2Kd are followed by the IRES-puromycin-resistance gene (PuroR)-polyadenylation signal sequence (pA). D, Analysis of the cell surface expression of MHC class I on ES-DC by flow cytometry. The staining patterns with specific Abs (thick lines) and isotype-matched controls (gray) are shown.

FIGURE 1.

Generation of ES-DC deficient in the intrinsic β2m gene and expressing recipient-matched MHC class I linked to β2m. A, Overview of the method for generation of ES-DC-expressing β2m-linked Kd, β2m+/−2m-Kd ES-DC and β2m−/−2m-Kd ES-DC. β2m−/− ES cells were generated from β2m+/− ES cells by high dose G418 selection. β2m+/− and β2m−/− ES cells were introduced with cDNA for the β2m-linked form of Kd and subsequently differentiated to generate β2m+/−/β2m-Kd ES-DC and β2m−/−/β2m-Kd ES-DC. B, The absence of wild-type β2m gene in the β2m−/− ES cell clone was confirmed by genomic PCR. C, Structure of the expression vector for β2m-linked H-2Kd (left) and a schematic representation of the encoded molecule (right). The β2m was fused to H-2Kd via a flexible 15 amino acid-long linker ([Gly4-Ser]3). The vector is driven by CAG promoter (pCAG) and cDNA for β2m-linked H-2Kd are followed by the IRES-puromycin-resistance gene (PuroR)-polyadenylation signal sequence (pA). D, Analysis of the cell surface expression of MHC class I on ES-DC by flow cytometry. The staining patterns with specific Abs (thick lines) and isotype-matched controls (gray) are shown.

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FIGURE 2.

Generation of TAP1-deficient, recipient-matched MHC class I gene-introduced ES-DC. A, Overview of the method for generation of Kd expressing, TAP1-deficient ES-DC. TAP1+/+ and TAP1−/− ES cells were transfected with cDNA for Kd and subsequently induced to differentiate into ES-DC. B, Structure of the mouse TAP1 genomic locus, the targeting construct and the mutant allele. Closed boxes indicate exons. The targeting construct contains a neomycin resistant gene (NeoR) and herpesvirus thymidine kinase gene (TK). The sets of PCR primers for the wild-type allele and mutant allele are shown as arrowheads. The position of the probe used for the Southern blot analysis is indicated as a small black box. The sizes of bands generated from the wild-type and mutant allele by digestion with XhoI (X) and EcoR V (E5) are indicated. The EcoR I (E1) restriction site is also shown. C, A genotype analysis of ES cells. Genomic DNA from ES cells was analyzed by genomic PCR and Southern blotting. D, Structure of the H-2Kd expression construct. The vector is driven by a CAG promoter (pCAG) and cDNA for H-2Kd are followed by the IRES-puromycin-resistance gene (PuroR)-polyadenylation signal sequence (pA). E, Surface phenotypes of genetically modified ES-DC. The surface expression of indicated MHC class I molecules on ES-DC were analyzed using flow cytometry. The expression of cell surface H-2Kd by TAP1−/−/Kd ES-DC were detected after cells were incubated with 10 μM H-2Kd-binding peptide (RSV M282–90) at 26°C for 12 h, subsequently incubated at 37°C for 4 h and stained with anti-H-2Kd Ab. The staining patterns with specific Abs (thick lines) and isotype-matched controls (gray) are shown.

FIGURE 2.

Generation of TAP1-deficient, recipient-matched MHC class I gene-introduced ES-DC. A, Overview of the method for generation of Kd expressing, TAP1-deficient ES-DC. TAP1+/+ and TAP1−/− ES cells were transfected with cDNA for Kd and subsequently induced to differentiate into ES-DC. B, Structure of the mouse TAP1 genomic locus, the targeting construct and the mutant allele. Closed boxes indicate exons. The targeting construct contains a neomycin resistant gene (NeoR) and herpesvirus thymidine kinase gene (TK). The sets of PCR primers for the wild-type allele and mutant allele are shown as arrowheads. The position of the probe used for the Southern blot analysis is indicated as a small black box. The sizes of bands generated from the wild-type and mutant allele by digestion with XhoI (X) and EcoR V (E5) are indicated. The EcoR I (E1) restriction site is also shown. C, A genotype analysis of ES cells. Genomic DNA from ES cells was analyzed by genomic PCR and Southern blotting. D, Structure of the H-2Kd expression construct. The vector is driven by a CAG promoter (pCAG) and cDNA for H-2Kd are followed by the IRES-puromycin-resistance gene (PuroR)-polyadenylation signal sequence (pA). E, Surface phenotypes of genetically modified ES-DC. The surface expression of indicated MHC class I molecules on ES-DC were analyzed using flow cytometry. The expression of cell surface H-2Kd by TAP1−/−/Kd ES-DC were detected after cells were incubated with 10 μM H-2Kd-binding peptide (RSV M282–90) at 26°C for 12 h, subsequently incubated at 37°C for 4 h and stained with anti-H-2Kd Ab. The staining patterns with specific Abs (thick lines) and isotype-matched controls (gray) are shown.

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β2m−/− ES cells were generated by the selection of the previously established β2m+/− ES cells (16) with high dose (1.5–2 mg/ml) of G418. Two of the 39 clones were found to have a β2m−/− genotype by genomic PCR (Fig. 1,B). Subsequently, an expression vector for β2m-Kd (Fig. 1,C) was introduced into both β2m+/− and β2m−/− ES cells to generate β2m+/−2m-Kd and β2m−/−2m-Kd ES cells, respectively. The genetically modified ES cell clones were subjected to differentiation culture to generate ES-DC. Theoretically, β2m-Kd is the only MHC class I molecule expressed on the cell surface of β2m−/−2m-Kd ES-DC. In the flow cytometric analysis, the cell surface expression of MHC class I molecules of H-2b haplotype, H-2Db and Kb, was detected in β2m+/−2m-Kd ES-DC but not in β2m−/−2m-Kd ES-DC. Expression of Kd was detected in the both types of ES-DC, as expected (Fig. 1 D).

To mutate the TAP1 gene in ES cells, the targeting vector (Fig. 2,B) was introduced into E14 ES cells to make several TAP1+/− ES cell clones. Subsequently, one of the TAP1+/− clones was subjected to selection with high dose of G418 as in the case of β2m−/− ES cells and TAP1−/− ES cell clones were isolated. Eight of the 88 surviving clones were found to be of TAP−/− genotype by genomic PCR (Fig. 2,C, left) and Southern blotting (Fig. 2,C, right). Next, an expression vector for Kd (Fig. 2,D) was introduced into both TAP1+/+ and TAP1−/− ES cells to generate TAP1+/+/Kd and TAP1−/−/Kd ES cells, respectively. Intrinsic MHC class I molecules, Db and Kb, as well as transgene-derived Kd, were not detected on the cell surface of TAP1−/−/Kd ES-DC (Fig. 2,E). A low level of cell surface expression of Kd on TAP1−/−/Kd ES-DC was observed after incubation of the ES-DC with Kd-binding peptide (RSV-M282–90) at 26°C for 12 h followed by the incubation at 37°C for 4 h (Fig. 2 E).

Collectively, ES-DC expressing only transgene-derived Kd but not intrinsic (H-2b haplotype-derived) MHC class I molecules on the cell surface were generated by the two methods of genetic modifications of ES cells.

H-2b MHC class I-reactive CD8+ T cells were prepared by a 5-day-culture of BALB/c (H-2d) spleen cells with irradiated 129 (H-2b) spleen cells. They produced IFN-γ in response to 129 mouse-derived BM-DC but not against BALB/c BM-DC, confirming that they responded specifically to H-2b MHC class I (Fig. 3,A, left panel). The four types of ES-DC derived from H-2b ES cells with genetic modification described above were cocultured with the H-2b-reactive CD8+ T cell line and the activation of the T cells was analyzed. Fig. 3 A shows that β2m+/−2m-Kd ES-DC (middle panel) and TAP1+/+/Kd ES-DC (right panel), expressing MHC class I of H-2b haplotype along with Kd, were recognized by the H-2 b-reactive CD8+ T cells, thus resulting in IFN-γ production at the magnitude similar to that observed in the case of 129 BM-DC. In contrast, H-2b-reactive CD8+ T cells showed practically no response to β2m−/−/β2m-Kd ES-DC or TAP1−/−/Kd ES-DC. These results indicate that ES-DC that were not recognized by alloreactive CD8+ T cells could be generated by the modification of β2m or TAP1 gene to inhibit surface expression of intrinsic MHC class I molecules.

FIGURE 3.

Reduced in vitro stimulation of alloreactive CD8+ T cells by β2m or TAP1 gene-deficient ES-DC. A, The response of H-2b-reactive CD8+ T cells to ES-DC was reduced by modification of the β2m (middle panel) or TAP1 (right panel) gene. To generate alloreactive (anti-H-2b) CTL, 5 × 106 BALB/c (H-2d) spleen cells were cultured with 2 × 106 irradiated 129 (H-2b) spleen cells for 5 days and after that the CD8+ cells were purified from the culture. The alloreactive CD8+ T cells (1 × 103) were cultured with the ES-DC (5 × 103) of the indicated genotype for 16 h. The 129 or BALB/c BM-DC were used as additional controls (left panel). The number of IFN-γ producing cells was measured using an ELISPOT assay. B, Allogeneic MHC class I-deficient ES-DC showed reduced potency to prime alloreactive CD8+ T cells in vitro. Splenocytes (5 × 106) derived from BALB/c mice were cultured with irradiated ES-DC (2 × 106) of the indicated genotype for 5 days and after that CD8+ T cells were purified from the culture. The IFN-γ producing response of the CD8+ T cells was measured with an ELISPOT with 129 BM-DC as stimulators. The results of the experiments with β2m deficient ES-DC (left panel) and TAP1-deficient ES-DC (right panel) are shown. Data are representative of at least two experiments with similar results. The data are the mean ± SD of triplicate assays. The asterisks indicate significant (p < 0.05, Student’s t test) differences between the two groups. ND, not detectable.

FIGURE 3.

Reduced in vitro stimulation of alloreactive CD8+ T cells by β2m or TAP1 gene-deficient ES-DC. A, The response of H-2b-reactive CD8+ T cells to ES-DC was reduced by modification of the β2m (middle panel) or TAP1 (right panel) gene. To generate alloreactive (anti-H-2b) CTL, 5 × 106 BALB/c (H-2d) spleen cells were cultured with 2 × 106 irradiated 129 (H-2b) spleen cells for 5 days and after that the CD8+ cells were purified from the culture. The alloreactive CD8+ T cells (1 × 103) were cultured with the ES-DC (5 × 103) of the indicated genotype for 16 h. The 129 or BALB/c BM-DC were used as additional controls (left panel). The number of IFN-γ producing cells was measured using an ELISPOT assay. B, Allogeneic MHC class I-deficient ES-DC showed reduced potency to prime alloreactive CD8+ T cells in vitro. Splenocytes (5 × 106) derived from BALB/c mice were cultured with irradiated ES-DC (2 × 106) of the indicated genotype for 5 days and after that CD8+ T cells were purified from the culture. The IFN-γ producing response of the CD8+ T cells was measured with an ELISPOT with 129 BM-DC as stimulators. The results of the experiments with β2m deficient ES-DC (left panel) and TAP1-deficient ES-DC (right panel) are shown. Data are representative of at least two experiments with similar results. The data are the mean ± SD of triplicate assays. The asterisks indicate significant (p < 0.05, Student’s t test) differences between the two groups. ND, not detectable.

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Next, the in vitro priming of alloreactive CD8+ T cells by ES-DC was examined. Spleen cells from BALB/c mice were cocultured with either of the four types of genetically engineered ES-DC for 5 days. After that, CD8+ T cells were isolated from the culture and their reactivity to 129-derived BM-DC (Fig. 3 B) was measured to assess the magnitude of priming in vitro of H-2b-reactive CD8+ T cells by the ES-DC. CD8+ T cells cultured with β2m+/−2m-Kd or TAP1+/+/Kd ES-DC in the induction phase responded to 129-derived BM-DC, indicating that both β2m+/−2m-Kd and TAP1+/+/Kd ES-DC primed H-2b-reactive CD8+ T cells. In contrast, CD8+ T cells cultured with β2m−/−/β2m-Kd or TAP1−/−/Kd ES-DC in the induction step exhibited reduced or no response to 129-derived BM-DC, thus indicating that the H-2b-reactive CD8+ T cells had not been well primed. These results suggest that the in vitro priming of allo-MHC class I-reactive T cells by ES-DC can be reduced through the genetic modification of β2m or TAP1.

The next experiments assessed whether or not priming of alloreactive CD8+ T cells upon in vivo administration of ES-DC could be avoided by the current strategy. The frequency of primed H-2b-reactive CD8+ T cells in mice was quantified by an ex vivo ELISPOT assay detecting the production of IFN-γ upon stimulation with 129-derived BM-DC. CD8+ T cells from naive BALB/c mice showed little response to 129-derived BM-DC. CD8+ T cells isolated from BALB/c mice injected 5 or 10 times with β2m+/−/β2m-Kd (Fig. 4,A) or TAP1+/+/Kd ES-DC (Fig. 4 B) clearly responded, thus indicating the priming of H-2b-reactive CD8+ T cells. The magnitude of the response of the mice injected ten times was lower than those injected five times, in both the β2m+/−2m-Kd ES-DC and TAP1+/+/Kd ES-DC-injected mice, thus suggesting that there is probably a limit in the frequency of alloreactive CD8+ T cells. In contrast, the frequency of H-2b-reactive CD8+ T cells in mice inoculated with β2m−/−2m-Kd or TAP1−/−/Kd ES-DC was very low, indicating that alloreactive CD8+ T cells were hardly primed in vivo by these ES-DC.

FIGURE 4.

Reduced in vivo stimulation of alloreactive CD8+ T cells by β2m or TAP1 gene-modified ES-DC. Splenic CD8+ T cells were isolated from naive BALB/c mice or those received multiple injections of β2m deficient (A) or TAP1 deficient (B) ES-DC (1 × 105/injection). Isolated CD8+ T cells (5 × 103) were cultured with BALB/c BM-DC (left panels) or 129 BM-DC (right panels) (5 × 103) for 16 h. The numbers of IFN-γ producing cells were measured using an ELISPOT assay. Data are representative of at least two experiments with similar results. The data are the mean ± SD of triplicate assays. Asterisks indicate significant (p < 0.05, Student’s t test) differences. ND, not detectable.

FIGURE 4.

Reduced in vivo stimulation of alloreactive CD8+ T cells by β2m or TAP1 gene-modified ES-DC. Splenic CD8+ T cells were isolated from naive BALB/c mice or those received multiple injections of β2m deficient (A) or TAP1 deficient (B) ES-DC (1 × 105/injection). Isolated CD8+ T cells (5 × 103) were cultured with BALB/c BM-DC (left panels) or 129 BM-DC (right panels) (5 × 103) for 16 h. The numbers of IFN-γ producing cells were measured using an ELISPOT assay. Data are representative of at least two experiments with similar results. The data are the mean ± SD of triplicate assays. Asterisks indicate significant (p < 0.05, Student’s t test) differences. ND, not detectable.

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According to a previous study by another group (23), BM-DC inoculated into allogeneic recipient mice are eliminated within a few days and the number of DC detected in the draining lymph node is lower than that of DC syngeneic to the recipient mice. In such circumstances, the rapid elimination of APC is mainly mediated by CD8+ T cells reactive to allogeneic MHC class I (24). As described so far, the β2m−/−2m-Kd ES-DC and TAP1−/−/Kd ES-DC did not express intrinsic H-2b-derived MHC class I molecule and escaped recognition by H-2b-reactive CD8+ T cells. Therefore, they were expected to have an advantage in surviving in allogeneic BALB/c mice, in comparison to ES-DC expressing H-2b gene encoded MHC class I on the cell surface.

To examine the effect of genetic modification on the survival of ES-DC upon injection into allogeneic mice, equal numbers of CMTMR-labeled ES-DC and CMFDA-labeled BALB/c derived BM-DC, as a control, were mixed and injected in the right forelimb footpad of BALB/c mice. After 48 h, a single cell suspension was made from the axillary and brachial lymph nodes and the fluorochrome-labeled DC were detected by using flow cytometry (Fig. 5). The number of β2m−/−2m-Kd ES-DC in the draining lymph nodes was ∼3 times higher than that of β2m+/−2m-Kd ES-DC. In the similar experiments, the number of detected TAP1−/−/Kd ES-DC was ∼4 times higher than that of TAP1+/+/Kd ES-DC. These results suggest that ES-DC without cell surface expression of intrinsic MHC class I molecule can thus escape elimination by alloreactive CTL.

FIGURE 5.

The survival advantage of β2m or TAP1 gene-deficient ES-DC in allogeneic recipient mice. BALB/c mice were injected s.c. into the forelimb with CMFDA-labeled BALB/c BM-DC (1 × 106) and CMTMR-labeled ES-DC (1 × 106). After 48 h, the mice were killed and the number of labeled BM-DC and ES-DC in the axillary and brachial lymph nodes was determined by flow cytometry. The number of detected ES-DC was normalized to control syngeneic BALB/c BM-DC [(CMTMR+ ES-DC/CMFDA+ BM-DC) × 100]. The results of experiments with β2m-deficient ES-DC (left) and TAP1-deficient ES-DC (right) are shown. Data are representative of two experiments with similar results. The data are the mean ± SD (5–6 mice per each group). Asterisks indicate significant (p < 0.05, Student’s t test) differences.

FIGURE 5.

The survival advantage of β2m or TAP1 gene-deficient ES-DC in allogeneic recipient mice. BALB/c mice were injected s.c. into the forelimb with CMFDA-labeled BALB/c BM-DC (1 × 106) and CMTMR-labeled ES-DC (1 × 106). After 48 h, the mice were killed and the number of labeled BM-DC and ES-DC in the axillary and brachial lymph nodes was determined by flow cytometry. The number of detected ES-DC was normalized to control syngeneic BALB/c BM-DC [(CMTMR+ ES-DC/CMFDA+ BM-DC) × 100]. The results of experiments with β2m-deficient ES-DC (left) and TAP1-deficient ES-DC (right) are shown. Data are representative of two experiments with similar results. The data are the mean ± SD (5–6 mice per each group). Asterisks indicate significant (p < 0.05, Student’s t test) differences.

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It has been noted that the CTL-mediated elimination of DC has a notable effect on the magnitude of immune responses in vivo (25) and the results so far described indicate that the β2m or TAP1 gene-modified ES-DC expressing only recipient-matched MHC class I could thus avoid elimination by CTL upon transfer into allogeneic recipients. Therefore, the ability of such ES-DC to elicit more robust Ag-specific immune responses in allogeneic recipients than ES-DC expressing intrinsic MHC class I was examined.

The priming of a RSV M2 protein epitope (M282–90)-specific and H-2Kd-restricted CTL by ES-DC administered into BALB/c mice was examined. M282–90 peptide-loaded ES-DC were injected i.p. into BALB/c mice twice with a 7-day interval. The spleen cells were isolated from the mice 7 days after the second injection and cultured in vitro in the presence of M282–90 peptide. After 6 days, the cultured spleen cells were recovered and assayed for their capacity to kill P815 mastcytoma cells (H-2d) prepulsed with the M2 peptide. M2 peptide-specific and Kd-restricted CTL was primed in BALB/c mice immunized with either of the 4 types of genetically modified ES-DC (Fig. 6 A). Therefore, ES-DC expressing Kd could prime Kd-restricted Ag-specific CTL, irrespective of cell surface expression of intrinsic MHC class I encoded by the H-2b haplotype.

FIGURE 6.

Priming of exogenous Ag-specific CTL by injection of genetically modified ES-DC loaded with antigenic peptide. A, RSV antigenic peptide-loaded ES-DC (1 × 105/injection/mouse) were injected i.p. into the naive mice twice with a 7-day interval. B and C, Non-Ag loaded ES-DC were injected 5 (B) or 10 (C) times with 7 days intervals before immunization with peptide-loaded ES-DC. The mice were sacrificed 7 days after the last injection of ES-DC and the spleen cells were isolated. The spleen cells were cultured in the presence of RSV peptide (1 μM) for 6 days and then analyzed on the RSV peptide-specific cytolytic activity by 5-h 51Cr release assay. As target cells, P815 cells either pulsed with M282–90 peptide (○) or control H-2Kd-restricted HIV gag p24199–207 peptide (•) were used. Data are representative of three experiments with similar results. The data are the mean specific lysis ± SD of triplicate assays. The asterisks indicate significant (p < 0.05, Student’s t test) differences.

FIGURE 6.

Priming of exogenous Ag-specific CTL by injection of genetically modified ES-DC loaded with antigenic peptide. A, RSV antigenic peptide-loaded ES-DC (1 × 105/injection/mouse) were injected i.p. into the naive mice twice with a 7-day interval. B and C, Non-Ag loaded ES-DC were injected 5 (B) or 10 (C) times with 7 days intervals before immunization with peptide-loaded ES-DC. The mice were sacrificed 7 days after the last injection of ES-DC and the spleen cells were isolated. The spleen cells were cultured in the presence of RSV peptide (1 μM) for 6 days and then analyzed on the RSV peptide-specific cytolytic activity by 5-h 51Cr release assay. As target cells, P815 cells either pulsed with M282–90 peptide (○) or control H-2Kd-restricted HIV gag p24199–207 peptide (•) were used. Data are representative of three experiments with similar results. The data are the mean specific lysis ± SD of triplicate assays. The asterisks indicate significant (p < 0.05, Student’s t test) differences.

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To determine whether or not ES-DC could prime M282–90 specific CTL in the presence of preprimed H-2b-reactive CTL, ES-DC without peptide loading were injected in BALB/c mice five (Fig. 6,B) or ten times (Fig. 6 C) with 7-day intervals and then the same ES-DC loaded with M282–90 peptide were injected. In addition, in this case, the specific CTL were primed by all of the four types of ES-DC. These results indicate that even in the presence of pre-activated alloreactive CTL, ES-DC expressing recipient-matched MHC class I are able to prime the Ag-specific CTL, whether or not the ES-DC express intrinsic MHC class I on their cell surface.

Next, antitumor immunity induced by the genetically modified ES-DC was assessed. To this end, a tumor cell line colon26/M2-Luc, a BALB/c-derived colon carcinoma cell line colon26 expressing RSV-M2 along with firefly luciferase, was generated. After the inoculation of the tumor cells, it was possible to quantify the number of cancer cells in mouse tissues by measuring the luciferase activity of tissue homogenates as reported (26). The luciferase activity in the homogenates of colon 26/M2-Luc cells was linearly correlated with the number of the cells in the range from 150 to 350,000 counts per second (Fig. 7 A). In a pilot study, when the tumor cells were injected into the mice i.p., most tumor cells were detected in the greater omentum and the mesenterium, and the luciferase activity of these two organs were in parallel (data not shown). Therefore, the luciferase activity of the greater omentum was chosen to be measured in the following studies.

FIGURE 7.

Induction of protective immunity by Ag-loaded genetically modified ES-DC against peritoneally disseminated tumor cells in allogeneic recipients. A, Homogenates were made from the indicated numbers of in vitro cultured colon26/M2-Luc cells and the luciferase activity was measured. r, correlation coefficient. B and C, BALB/c mice were injected with M282–90 peptide- or control HIV p24199–207 peptide-loaded ES-DC (1 × 105/mouse) i.p. on day –7 and challenged with colon26/M2-Luc (1 × 106/mouse) i.p. on day 0. On day 10, the mice were sacrificed and luciferase activity of the greater omentum was measured. Luciferase activity of tissue lysates was converted to tumor cell number in the greater omentum based on the standard curve shown in A. The results of mice treated with β2m+/−2m-Kd ES-DC or β2m−/−2m-Kd ES-DC are shown in B. The results of mice treated with TAP1+/+/Kd ES-DC or TAP1−/−/Kd ES-DC are shown in C. All data are representative of at least two experiments with similar results. Values for individual mice injected with M282–90 peptide-loaded ES-DC (○) and HIV p24199–207 peptide-loaded ES-DC (•) are shown; bars indicate median values. The asterisks indicate significant (p < 0.05) differences between two groups based on the Mann-Whitney U test. NS, not significant.

FIGURE 7.

Induction of protective immunity by Ag-loaded genetically modified ES-DC against peritoneally disseminated tumor cells in allogeneic recipients. A, Homogenates were made from the indicated numbers of in vitro cultured colon26/M2-Luc cells and the luciferase activity was measured. r, correlation coefficient. B and C, BALB/c mice were injected with M282–90 peptide- or control HIV p24199–207 peptide-loaded ES-DC (1 × 105/mouse) i.p. on day –7 and challenged with colon26/M2-Luc (1 × 106/mouse) i.p. on day 0. On day 10, the mice were sacrificed and luciferase activity of the greater omentum was measured. Luciferase activity of tissue lysates was converted to tumor cell number in the greater omentum based on the standard curve shown in A. The results of mice treated with β2m+/−2m-Kd ES-DC or β2m−/−2m-Kd ES-DC are shown in B. The results of mice treated with TAP1+/+/Kd ES-DC or TAP1−/−/Kd ES-DC are shown in C. All data are representative of at least two experiments with similar results. Values for individual mice injected with M282–90 peptide-loaded ES-DC (○) and HIV p24199–207 peptide-loaded ES-DC (•) are shown; bars indicate median values. The asterisks indicate significant (p < 0.05) differences between two groups based on the Mann-Whitney U test. NS, not significant.

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BALB/c mice were injected i.p. with ES-DC loaded with M282–90 peptide and other mice were injected with ES-DC prepulsed with irrelevant control peptide (HIV gag p24199–207) with Kd-binding affinity. One week after the ES-DC injection, the mice were challenged i.p. with colon26/M2-Luc. After 10 days, the growth of tumor cells was evaluated by measuring the luciferase activity in the homogenate of the greater omentum. In the mice injected with either of the β2m−/−/β2m-Kd ES-DC (Fig. 7,B) or TAP1−/−/Kd ES-DC (Fig. 7 C) loaded with M2 peptide, tumor growth was significantly reduced in comparison to the mice injected with the same ES-DC loaded with the control peptide. Therefore, the two types of genetically modified ES-DC could induce Ag-specific antitumor immunity. The antitumor effect induced by β2m−/−2m-Kd ES-DC was significantly stronger than that induced by β2m+/−2m-Kd ES-DC, indicating that the disruption of intrinsic β2m gene and introduction of β2m-linked MHC class I in ES-DC may provide an advantage in the induction of Ag-specific antitumor immunity.

To induce T cell-mediated anticancer immunity, vaccination with DC loaded with tumor Ag-derived peptides or tumor cell lysates are being clinically tested (27, 28). For such purposes, DC are generated from monocytes obtained from peripheral blood of the patients. However, monocytes are not easily propagated in vitro and apheresis, a procedure sometimes invasive for the patients, is necessary to obtain a sufficient number of monocytes as the source of DC. In addition, the culture to generate DC should be done separately for each patient and for each treatment and thus the presently used method is too labor-intensive and costly to be broadly applied.

ES cells exhibit the remarkable properties of self-renewal and pluripotency. This capacity allows for the production of sizeable quantities of therapeutic cells of the hematologic lineage, including DC (29). If the ES-DC method is clinically applied, it will be possible to generate genetically engineered DC-expressing target Ags or immunostimulatory molecules, without use of virus-based vectors. However, considering the medical application, one drawback of the ES-DC method is the unavailability of human ES cells genetically identical to the patients to be treated (12). Specifically, an HLA allele mismatch between ES cells and patients is a crucial problem.

In previous studies, it was shown that allogeneic BM-DC are rapidly eliminated from the draining lymph nodes during the course of a primary alloreactive responses (23, 30). Another study revealed that the elimination of transferred APC in allogeneic recipients is mainly mediated by T cells reactive to allogeneic MHC class I but not MHC class II (24). Therefore, if the expression of the intrinsic MHC class I by ES-DC could be blocked, then ES-DC inoculated into allogeneic recipients would escape elimination by the alloreactive T cells of the recipients. However, the genomic region including the MHC class I genes spans more than 1,000 kb in both the mouse and human genome and complete elimination of such a large genomic region by gene-targeting technique is currently infeasible. The feasible candidates for genetic modification are the genes that encode β2m and TAP, which regulate MHC class I expression (31, 32). Therefore, the present study adopted a strategy to block the cell surface expression of the MHC class I molecules by elimination of the β2m or TAP1 gene.

Both alleles of the TAP1 or β2m gene were disrupted in 129-derived ES cells (H-2b) and subsequently expression vectors for the recipient (BALB/c)-matched Kd or β2m-linked form of Kd were introduced. The genetically modified ES cells were subjected to the differentiation culture to generate TAP1−/−/Kd ES-DC and β2m−/−2m-Kd ES-DC. As intended, the β2m−/−2m-Kd ES-DC expressed only Kd molecule as MHC class I molecules on the cell surface. TAP1−/−/Kd ES-DC hardly expressed any classical MHC class I and a low level of cell surface expression of Kd was observed after incubation with Kd-binding peptide. In vitro, β2m−/−2m-Kd and TAP1−/−/Kd ES-DC were not recognized by pre-activated H-2b-reactive CD8 T+ cells and the ES-DC did not prime H-2b-reactive CD8+ T cells (Fig. 3). When these cells were inoculated into BALB/c mice, they did not prime H-2b reactive CD8+ T cells in vivo (Fig. 4).

Consistent with these results, β2m−/−/β2m-Kd ES-DC and TAP1−/−/Kd ES-DC had a survival advantage in comparison to β2m+/−2m-Kd ES-DC and TAP1+/+/Kd ES-DC, when transferred into BALB/c mice (Fig. 5). The results suggest that ES-DC deficient in β2m or TAP1 and expressing only recipient-matched MHC class I were resistant to elimination by alloreactive CTL. It has been shown that CTL-mediated elimination of DC has a notable effect on the magnitude of immune responses in vivo (25, 33). Therefore, β2m- or TAP1-deficient ES-DC should be able to elicit more robust priming of Ag-specific CTL in allogeneic recipients than ES-DC expressing intrinsic MHC class I. When loaded with RSV-derived peptide and inoculated into BALB/c mice, not only β2m−/−2m-Kd and TAP1−/−/Kd ES-DC but also β2m+/−2m-Kd and TAP1+/+/Kd ES-DC primed Kd-restricted, RSV peptide-specific CTL (Fig. 6). Unexpectedly, there was no significant difference in the magnitude of priming of Ag-specific CTL among these ES-DC. CTL-mediated allogeneic DC elimination is mainly dependent on the perforin/granzyme B pathway (24, 30). Therefore, the result shown in Fig. 6 may be due to resistance of ES-DC to killing by CTL that is attributed partly to the high level of expression of SPI-6, the granzyme B-specific protease inhibitor, in ES-DC (34). In addition, in the experiments shown in Fig. 6, we cultured spleen cells isolated from immunized mice for 5 days in the presence of RSV-M2 peptide to amplify RSV-specific CTL before the cytotoxicity assay, because we could not detect RSV-specific CTL activity in a direct ex vivo killing assay. Probably, in the data shown in Fig. 6, difference in the CTL activity induced in vivo by the different genotype of ES-DC may have been masked by this culture procedure.

In the present study, the MHC class II haplotype of ES-DC was always b while that of the recipient mice (BALB/c) was d. Therefore, there was mismatch in MHC class II haplotype between ES-DC and the recipient mice in all of the experiments. Because the mismatch of the MHC class II allele has been reported to not cause any acute elimination of transferred APC (24), the class II mismatch may not have negatively affected the priming of Ag-specific CTL in the present study. Instead, alloreactive helper T cells are expected to enhance the CTL response via cytokine production, although we did not experimentally address this issue in the present study.

Theoretically, the issue of histocompatibility related to the ES cell-based medical technology may be resolved by the recent development of induced pluripotent stem (iPS) cells that can be generated by introduction of several defined genes into somatic cells (35, 36, 37, 38). However, the medical application of iPS cells nevertheless has some drawbacks. The use of virus vectors is necessary to generate iPS cells and generation of iPS cells for individual patients may be too costly, time consuming, and labor-intensive to be broadly applied. The genetic modification of ES cells or iPS cells to modify cell surface HLA class I by the presently reported methods may be more economical, faster and thus, more realistic than the individual generation of “fully personalized iPS cells”.

Although targeted gene disruption of OCT4 and HPRT in human ES cells has been reported (39), the methodology of gene targeting for human ES cells has not been well established at present. Therefore, as an alternative strategy, we are planning to generate iPS cells from patients with Type I bare lymphocyte syndrome caused by mutation of the TAP 1 or TAP 2 gene (40, 41). Once a clone of TAP- or β2m-deficient human ES or iPS cells is established, a premade library of pluripotent stem cell clones expressing various types of HLA class I can be generated by the introduction of various HLA class I genes. Such a pluripotent stem cell bank may serve as a source of not only DC but also of various kinds of differentiated cells that may be useful in the field of regenerative medicine.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by Grants-in-Aid 16590988, 17390292, 17015035, 18014023, 19591172, and 19059012 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan; the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases launched as a project commissioned by MEXT, Japan; Research Grant for Intractable Diseases from Ministry of Health and Welfare, Japan; and grants from Japan Science and Technology Agency (JST), the Uehara Memorial Foundation, and the Takeda Science Foundation.

3

Abbreviations used in this paper: DC, dendritic cell; ES cell, embryonic stem cell; ES-DC, embryonic stem cell-derived DC; BM-DC, bone marrow-derived DC; β2-microglobulin, β2m; HA, hemagglutinin; RSV, respiratory syncytical virus; PEF, primary mouse embryonic fibroblast; CMTR, chloromethyl-benzoyl-amino-tetramethyl-rhodamine; CMFDA, chlorometylfluorescein diacetate; iPS cell, induced pluripotent stem cell; Luc, luciferase; IRES, Internal ribosomal entry site.

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