1. Volume 29, Issue 1, Pages 162-175.e9 (October 2019)
Type I Interferon Delivery by iPSC-Derived Myeloid Cells Elicits Antitumor Immunity via XCR1+ Dendritic Cells 
Nobuhiro Tsuchiya, Rong Zhang, Tatsuaki Iwama, Norihiro Ueda, Tianyi Liu, Minako Tatsumi, Yutaka Sasaki, Ranmaru Shimoda, Yuki Osako, Yu Sawada, Yosuke Kubo, Azusa Miyashita, Satoshi Fukushima, Zhao Cheng, Ryo Nakaki, Keiyo Takubo, Seiji Okada, Shin Kaneko, Hironobu Ihn, Tsuneyasu Kaisho, Yasuharu Nishimura, Satoru Senju, Itaru Endo, Tetsuya Nakatsura, Yasushi Uemura 
Cell Reports 
Volume 29, Issue 1, Pages e9 (October 2019)
DOI: /j.celrep
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2. Cell Reports 2019 29, 162-175.e9DOI: (10.1016/j.celrep.2019.08.086)
Copyright © 2019 The Authors Terms and Conditions

3. Figure 1
The iPSC-Derived Proliferating Myeloid Cells Genetically Engineered to Express Type I IFN
(A) Morphology of pMCs and IFN-α-pMCs. Upper panels, phase-contrast images of the indicated cells. Lower panels, May-Giemsa staining of cytospin cell specimens. Scale bar, 10 μm.
(B) Representative flow cytometry profiles of the indicated surface molecules.
(C) Cell proliferation. IFN-α-pMCs (2.0 × 103 cells/well) were seeded in 96-well culture plates in the presence of indicated cytokines. Proliferation was determined at each time point.
(D) IFN-α production. pMCs or IFN-α-pMCs (5.0 × 104 cells/well) were seeded in 6-well culture plates and cultured for 24 h in the presence of indicated cytokines.
(E) Gene expression signature of pMCs and IFN-α-pMCs. Heatmap shows differentially expressed genes (log-fold change ≥ 1 or ≤ −1) between pMCs and IFN-α-pMCs determined using RNA sequencing (RNA-seq) data. Normalized Z score values were calculated for each differentially expressed gene (high, red; low, blue). The distribution of the expression level of the differentially expressed genes (DEGs) is shown on the color key legend (top left). The Venn diagram represents the upregulated genes classified among type I–III ISGs using the Interferome database. See also Table S1.
(F) pMCs, IFN-α-pMCs, or Ifnar1-disrupted IFN-α-pMCs (IFN-α-pMC-Ifnar1−) were cultured for 16 h without adding cytokines. Irf7 and Cxcl10 mRNA levels were quantified using qRT-PCR. See also Figures S1A and S1B.
(G) CXCL10 production. pMCs or IFN-α-pMCs (2.0 × 106 cells/well) were cultured for 24 h in the presence of the indicated antibody (20 μg/mL).
(A–D, F, and G) Representative data from two independent experiments are presented. (C, D, F, and G) Shown are means ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01.
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4. Figure 2
Local Administration of IFN-α-iPSC-pMCs Controls Distant Tumors
(A–D) Upper panels, schema for tumor implantation and treatment.
(A–C) C57BL/6 mice were s.c. inoculated with B16-F10-OVA cells in both hindlimbs (A), B16-F10-OVA cells in the right hindlimb and left flank (B), or MC38 cells in the bilateral hindlimbs (C) and left untreated or treated peritumorally (right hindlimb) with either pMC or IFN-α-pMCs at indicated time points.
(D) BALB/c mice were s.c. inoculated with CT26 cells in both hindlimbs and treated as in (A).
(E) Kinetics of serum IFN-α. C57BL/6 mice were s.c. inoculated with B16-F10-OVA cells in both hindlimbs and treated peritumorally (right hindlimb) with IFN-α-pMCs (1.0 × 106 cells) or rIFN-α (104 units) on days 5, 6, and 7. At 0.5, 1, 3, 6, and 24 h after last treatment, serum IFN-α levels were evaluated using enzyme-linked immunosorbent assay. Representative data from two independent experiments are presented. Shown are means ± SD (n = 3).
(F) C57BL/6 mice were s.c. inoculated with B16-F10-OVA cells in both hindlimbs and left untreated or treated peritumorally (right hindlimb) with IFN-α-pMCs (1.0 × 106 cells) or rIFN-α (104 units) at the same time point as in (A).
(G) WT mice were inoculated with Ifnar1-disrupted B16-F10-OVA (B16-F10-OVA-Ifnar1−) cells. See also Figures S2G–S2K.
(H–K) Ifnar1-deficient (Ifnar1−/−) mice (H), Ifnar1−/−-bone marrow (BM)-chimeric mice (WT host) (I), WT-BM-chimeric mice (Ifnar1−/− host) (J), or WT-BM-chimeric mice (WT host) (K) were inoculated with WT B16-F10-OVA cells.
(L) Ifnar1−/− mice were inoculated with B16-F10-OVA-Ifnar1− cells. See also Figure S2L.
(A–D and F–L) Tumor volume is shown as the mean ± SEM of two independent experiments (n = 8–12 mice). ∗p < 0.05, ∗∗p < 0.01.
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5. Figure 3
IFN-α-iPSC-pMCs Propagate the Type I IFN-Associated Molecular Signature and Induce CD8+ T Cell-Mediated Antitumor Immunity
(A) C57BL/6 mice were s.c. inoculated with B16-F10-OVA cells in both hindlimbs; treated peritumorally (right hindlimb) with IFN-α-pMCs, as in Figure 2A; and i.p. injected with anti-CD4, anti-CD8, or anti-NK1.1 depletion antibody or control antibody twice weekly. Tumor volume (on day 18) of left (distant) and right (treated) tumors are shown as the mean ± SEM of two independent experiments (n = 10 mice). See also Figures S3A and S3B.
(B) B16-F10-OVA tumor-bearing C57BL/6 mice were treated as in Figure 2A, and bilateral tumor tissue gene expression was analyzed using RNA-seq. The overlapping genes between those differentially expressed in untreated versus IFN-α-pMC-treated tumor or pMC-treated versus IFN-α-pMC-treated tumor, for distant or treated tumors, respectively, were identified. Of these, upregulated ISGs in IFN-α-pMC-treated tumors were classified as type I–III ISGs using the Interferome database. Left Venn diagram, distant tumor; right Venn diagram, treated tumor. See also Figures S3C–S3G and Tables S2–S5.
(C) mRNA levels of indicated genes in tumor tissues quantified using qRT-PCR. Shown are the mean ± SEM of two independent experiments (n = 6). See also Figure S3H.
(D and E) B16-F10-OVA tumor-bearing C57BL/6 mice were treated as in Figure 2A. On day 14, distant tumor tissues were analyzed for (D) H-2Kb/OVA-tetramer+ cell (CD3+CD8+ gated) or (E) perforin+ cell (CD3+CD8+ gated) frequency and number. Representative data from two independent experiments are presented. Shown are means ± SD (n = 3). See also Figures S3I–S3K and Tables S6 and S7.
(F and G) B16-F10-OVA tumor-bearing mice were treated as in Figure 2A and i.p. injected with FTY720 at indicated time points (F) or anti-CXCR3 blocking antibody or control antibody twice weekly (G). Tumor volume is presented as the mean ± SEM of two independent experiments (n = 8–10 mice). ∗p < 0.05, ∗∗p < 0.01.
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6. Figure 4 IFN-α-iPSC-pMCs Enhance Host XCR1+ DC Cross-Presentation
(A and B) Mice were s.c. inoculated with B16-F10-OVA cells in the right hindlimb and left untreated or treated peritumorally (right hindlimb) with pMCs or IFN-α-pMCs on day 5. At 24 h after treatment, TdLN cells were harvested, and surface H-2Kb/OVA (A) and CD86 (B) expression on indicated subsets was assessed using flow cytometry. Upper panels, representative histograms of two independent experiments. Bar graphs (lower panels) show mean fluorescence intensity (MFI). Representative data from two independent experiments are presented. Shown are means ± SD (n = 3). See also Figure S4A.
(C) B16-F10-OVA tumor-bearing DTX-treated Xcr1-DTR+/− or WT littermate mice were treated as in (A) and adoptively transferred with eFluor 670-labeled GFP+ OT-I T cells on day 6. TdLNs were harvested 4 days after intravenous injection of OVA-specific T cells, and their proliferation was assessed based on eFluor 670 dilution by flow cytometry. Bar graph (upper panel) shows the mean ± SEM of two independent experiments (n = 7 mice). Lower panel, representative histogram of two independent experiments (IFN-α-pMC treatment).
(D–F) B16-F10-OVA tumor-bearing mice were treated as in Figure 2A.
(D) DTX-treated Itgax-DTR+/− bone marrow chimeric mice.
(E) DTX-treated WT littermate and Xcr1-DTR+/− mice.
(F) Ifnar1flox/flox (WT), Itgax-Cre × Ifnar1flox/flox, and Cd4-Cre × Ifnar1flox/flox mice. Tumor volume is shown as the mean ± SEM of two independent experiments (n = 10–14 mice). ∗p < 0.05, ∗∗p < See also Figure S4D.
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7. Figure 5
Host STING Pathway-Independent Therapeutic Efficacy of IFN-α-iPSC-pMCs
B16-F10-OVA tumor-bearing mice were treated as in Figure 2A. (A) Sting−/−, (B) Irf3−/−, and (C) Irf7−/− mice. Tumor volume is shown as the mean ± SEM of two independent experiments (n = 10–12 mice). ∗p < 0.05, ∗∗p < See also Figure S5.
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8. Figure 6
IFN-α-iPSC-pMC and PD-1/PD-L1 Blockade Combination Synergistically Inhibits Tumor Growth
(A) B16-F10-OVA tumor-bearing C57BL/6 mice were treated as in Figure 2A. The mRNA levels of Cd274 (Pd-l1) were quantified using qRT-PCR. Shown are the mean ± SEM of two independent experiments (n = 6). See also Figure S6A.
(B) Sections of distant tumor tissue or TdLN from IFN-α-pMC-treated mice (day 11) were hybridized with antisense Cd274 RNA probes (purple) and stained with anti-CD8 monoclonal antibody (mAb) (brown). Squares in the left panels are magnified and presented in the right panels. See also Figures S6B and S6C.
(C) B16-F10-OVA tumor-bearing C57BL/6 mice were left untreated or treated with IFN-α-pMC alone, anti-PD-L1 antibody alone, or in combination.
(D and E) B16-F10-OVA tumor-bearing WT mice were treated as in (C). On day 14, tumor tissues were analyzed for the number of (D) H-2Kb/OVA-tetramer+ cells (CD3+CD8+ gated) or (E) perforin+ cells (CD3+CD8+ gated). Representative data from two independent experiments are presented. Shown are means ± SD (n = 3). See also Figure S6D.
(F) Five weeks after complete tumor rejection, mice administered combination therapy or naive mice were inoculated with B16-F10-OVA cells (2 × 106 cells) on the left flank. Tumor volume is shown as mean ± SD (n = 4).
(G) Kaplan-Meier survival curve of rechallenged or naive mice (n = 4). The mice were monitored for tumor growth and survival until the mice died or were sacrificed when the tumors exceeded 20 mm in diameter. Data are representative of two independent experiments. ∗p < 0.05, ∗∗p < 0.01 by the log-rank test.
(H) Spleen CD8+ T cells isolated from mice administered combination therapy (after 35 days of complete tumor rejection) or naive mice were stimulated with OVA peptide- or control SIY peptide (SIYRYYGL)-loaded, irradiated RMA-S for 24 h. The frequency of IFN-γ-producing cells was assessed using ELISPOT assay. EL4 and B16-F10-OVA cells served as references. Shown are means ± SD (n = 6).
(I–K) Tumor-bearing mice were left untreated or treated with IFN-α-pMC alone (right), anti-PD-L1 antibody alone, or in combination.
(I) C57BL/6 mice were inoculated with MC38 cells.
(J) BALB/c mice were inoculated with CT26 cells.
(K) BALB/c mice were inoculated with 4T1 cells.
(F and H) Representative data from two independent experiments are shown. (C, I, J, and K) Tumor volume is shown as the mean ± SEM of two independent experiments (n = 10–12 mice). ∗p < 0.05, ∗∗p < See also Figures S6E–S6G.
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9. Figure 7
Administration of IFN-α-iPSC-pMCs Does Not Affect Hematopoiesis
B220+, CD11b+, and Gr-1+ cell frequency in the peripheral blood CD45.1+ or CD45.2+ cells was analyzed using flow cytometry at 12 weeks after transplantation. Shown are the means ± SEM of two independent experiments (n = 8). ∗p < 0.05, ∗∗p < See also Figures S7A–S7C.
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