Research ArticleDENDRITIC CELLS

Direct reprogramming of fibroblasts into antigen-presenting dendritic cells

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Science Immunology  07 Dec 2018:
Vol. 3, Issue 30, eaau4292
DOI: 10.1126/sciimmunol.aau4292
  • Fig. 1 Screening for DC-inducing factors identifies PU.1, IRF8, and BATF3 (PIB) combination.

    (A) Experimental design to screen DC-inducing transcription factors (TFs). Clec9a-tdTomato (Clec9a-tdT) double transgenic mouse embryonic fibroblasts (MEFs) were cotransduced with lentiviral particles encoding candidate TFs and M2rtTA and cultured in the presence of doxycycline (Dox) and monitored for tdT (red) expression. (B) MEFs transduced with M2rtTA, PU.1 + C/EBPα, or PU.1 + IRF8 + BATF3 (PIB) were analyzed by fluorescent microscopy and flow cytometry 5 days after the addition of Dox. Scale bar, 200 μm. (C) Quantification of tdT+ cells after removal of individual TFs from the pool or their individual expression at day 5 (n = 3, mean ± SD). (D and E) Quantification of tdT+ cells after transduction with PIB combined with (D) individual TFs from the 18 candidates or (E) hematopoietic TFs at day 8 (n = 2 to 6, mean ± SD). (F and G) Kinetics of Clec9a-tdT reporter activation analyzed by (F) flow cytometry or (G) time-lapse microscopy. White arrowheads indicate an emerging tdT+ cell. Scale bar, 200 μm. (H) Immunofluorescence for F-actin (green) and DAPI (blue) highlighting tdT+ cell morphology. Scale bars, 50 μm. (I) Scanning electron microscopy analysis of tdT+ and M2rtTA-transduced cells. Scale bars, 10 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Bonferroni’s test.

  • Fig. 2 Induced DCs express classical APC surface molecules and cDC1 receptors.

    (A) Flow cytometry analysis of CD103 and XCR1 in tdT+ and tdT populations of PIB-transduced MEFs 8 days after the addition of Dox. (B) Quantification of CD8α, CD4, CD11b, B220, F4/80, CD64, and SIRPα expression in tdT+ and tdT populations (n = 2, line indicates mean). (C) MHC-I and MHC-II expression in MEFs transduced with M2rtTA and PIB at day 7. (D) Kinetics of MHC-II surface expression. (E) Quantification of MHC-II+ cells after removal of individual transcription factors from PIB at day 5 (n = 3 to 4, mean ± SD). (F) Quantification of MHC-II+ cells at day 5 within tdT+ population after transduction with PIB, PIB and IRF4, or upon their individual removal (n = 2 to 3, mean ± SD). (G) CD80 and CD86 expression in tdT+ MHC-II+ and tdT+ MHC-II populations at day 5. (H) CD40 and MHC-II expression at day 7 before or (I) after overnight LPS stimulation. CD80 expression in MHC-II+ CD40+ (red) and MHC-II CD40+ (blue) populations. *P < 0.05, **P < 0.01, one-way ANOVA with Bonferroni’s test.

  • Fig. 3 PIB induce global cDC1-like gene expression program.

    (A) PIB-transduced MEFs were FACS sorted and profiled using full-length transcript single-cell mRNA-seq at day 3 (tdT+), day 7 (tdT+), and day 9 (tdT+ MHC-II+). MEF and splenic cDC1 cells (CD11c+ MHC-II+ CD8α+) were used as controls. (B) t-SNE analysis of genome-wide transcriptomes showing clustering of 163 single cells. Each dot represents an individual cell (27 MEFs; 16 day 3, 30 day 7, and 29 day 9 iDCs; and 61 cDC1s). (C) Heat map showing expression of the 6525 most variable genes and four gene clusters. (D) Expression levels of fibroblast-associated genes shown as Census counts median values ± 95% confidence interval. (E) Violin plots showing expression distribution of genes in clusters II, III, and IV. Log values of Census counts are shown; horizontal lines correspond to median values. (F) Top five GO biological processes (left) and cellular components (right). (G) Cumulative median expression levels of cDC1 and cDC2 gene signatures. (H) Violin plots showing expression distribution of DC transcriptional regulators. (I) Total (left) and endogenous (right) expression of Spi1, Irf8, and Batf3 are shown as log counts presented as box plots.

  • Fig. 4 Reconstruction of single-cell reprogramming trajectory highlights different maturation states of iDCs.

    (A) Pseudotime ordering of single cells (line) in a two-dimensional independent component space during reprogramming. MEF; iDC at day 3, day 7, and day 9; and cDC1 are shown. (B) Individual cells are colored by cell state. The number of cells in each cell state is depicted inside parentheses. (C) Top five GO biological processes (BP), mouse phenotypes, and pathway enrichment analysis of genes differentially expressed between state 2 and state 3. (D) Gene expression levels of Ciita, H2-Eb1, Lgmn, and Tnfrsf1a in single cells from the three cell states. (E) GSEA between day 9 iDCs and cDC1 was performed against the immunologic signatures collection. Gene sets were ordered by normalized enrichment score (NES). False discovery rate (FDR) q values are shown (<0.25). Black lines represent DC gene sets. Bottom right panel shows enrichment plots for IFN-stimulated DC gene sets. (F) Top five GO BP, cellular components (CC), and pathway enrichment analysis of genes differentially expressed between day 9 iDC and cDC1. (G and H) Violin plots showing expression distribution of (G) the maturation regulators Stat1 and Stat6 and (H) Ciita with corresponding normalized promoter usage in day 9 iDC and cDC1 (right).

  • Fig. 5 Induced DCs are functional APCs.

    (A) Cytokine secretion of sorted tdT+ cells after stimulation with LPS or PIC and (B) IL12p40 expression after stimulation with LPS/profilin/CD40L. (C) Purified tdT+ cells were incubated with CellVue far red–labeled dead cells or (D) DAPI-labeled dead cells and analyzed by time-lapse microscopy for 12.5 hours. Scale bar, 50 μm. (E and F) iDCs at day 8 and splenic CD11c+ MHC-II+ (cDC) were cocultured with CFSE-labeled OT-II Rag2KO CD4+ T cells. CD44 activation and CFSE dilution were quantified (n = 2 to 4, mean ± SD). (G and H) β-Lactamase’s export to cytosol of iDCs at day 7 (gated in tdT+) and BM-DCs measured as CCF4 cleavage. (I) iDCs at day 16 were cocultured with B3Z T cell hybridoma for 18 hours with increasing concentrations of OVA protein (left) and LPS or PIC stimulation (middle). Purified tdT+ cells at day 8 and BM-DCs were pulsed with OVA for 10 hours before coculture with B3Z in the presence of PIC (right). (J and K) MEFs, purified tdT+ and tdT cells at day 8, BM-DCs, and cDC1 were pulsed with OVA for 10 hours before coculture with CTV-labeled OT-I CD8+ T cells. CD44 activation and CTV dilution were quantified (n = 3, mean ± SD).

  • Fig. 6 Polycistronic vectors encoding PIB increase reprogramming efficiency in mouse embryonic and adult fibroblasts.

    (A) Schematic representation of the two polycistronic (poly) lentiviral plasmids encoding Spi1, Irf8, and Batf3 in different orders separated by P2A and T2A self-cleaving peptides. (B) Flow cytometry quantification of tdT+ cells after transduction with PIB individual vectors (P + I + B) and PIBpoly and IPBpoly vectors at day 8 after the addition of Dox (n = 7, mean ± SD). (C) Quantification of MHC-II+ cells in tdT+ and tdT populations (n = 6, mean ± SD). (D) Western blot analysis of PU.1 and IRF8 protein levels in MEFs transduced with PIBpoly and IPBpoly plasmids at day 2. M2rtTA (M2)–transduced cells and calnexin (CANX) levels were included as controls. (E to G) MHC-II and CD45 expression in (E) PIBpoly-transduced Clec9a-tdT MEFs, (F) C57BL/6 MEFs, and (G) adult tail-tip fibroblasts (TTFs) at day 9. ****P < 0.0001, one-way ANOVA with Bonferroni’s test.

  • Fig. 7 PIB induce reprogramming of human embryonic and adult fibroblasts to DC-like cells.

    (A) Strategy to generate human iDCs (hiDCs) upon transduction of fibroblasts with the PIBpoly vector. (B) Bright-field microscopy (left) and scanning electron microscopy (SEM; right) of PIBpoly-transduced human embryonic fibroblasts (HEFs) at day 9 after the addition of Dox. Scale bars, 50 μm (left) and 10 μm (right). (C and D) Kinetics of CD45 and HLA-DR expression of PIBpoly- and M2rtTA-transduced HEFs analyzed by flow cytometry (n = 2 to 5, mean ± SD). (E) CD141 and CLEC9A expression in HLA-DR+ cells at day 9. Fluorescence minus one (FMO) was included as control. (F) Incorporation of Alexa Fluor 647–labeled OVA (OVA-A647) by PIBpoly-transduced HEFs after 20-min incubation at 37° or 4°C. (G) PIBpoly-transduced HEFs were incubated overnight with CellVue far red–labeled dead cells at day 8. HLA-DR and HLA-DR+ populations are shown. (H) Bright-field microscopy of PIBpoly-transduced human dermal fibroblasts (HDFs) at day 9. (I) CD45 and HLA-DR expression of PIB- and M2rtTA-transduced HDFs at day 9. (J) CD141 and CLEC9A expression in HLA-DR+ cells. Scale bars, 100 μm.

Supplementary Materials

  • Supplementary material for this article is available at immunology.sciencemag.org/cgi/content/full/3/30/eaau4292/DC1

    Methods

    Fig. S1. Candidate transcription factors to instruct DC cell fate.

    Fig. S2. Clec9a-based reporter to identify transcription factors to induce DC fate.

    Fig. S3. Screening for transcription factors to activate Clec9a-tdT.

    Fig. S4. DC morphology and dendrites are established in mouse fibroblasts.

    Fig. S5. Spi1, Irf8, and Batf3 are enriched in cDC1 cells.

    Fig. S6. Global single-cell gene expression analysis during iDC reprogramming.

    Fig. S7. Stepwise transitions during iDC reprogramming.

    Fig. S8. iDC reprogramming does not induce alterations associated with tumorigenesis.

    Fig. S9. Analysis of iDC maturation states.

    Fig. S10. Induced DCs are functional APCs.

    Fig. S11. Polycistronic vector induces cDC1-like transcriptional program.

    Fig. S12. PIB induce human iDC reprogramming.

    Fig. S13. FACS gating strategies.

    Table S1. Analysis of candidate transcription factors.

    Table S2. Single-cell mRNA-seq data analysis.

    Table S3. Pseudotime single-cell mRNA-seq data analysis.

    Table S4. Population mRNA-seq data analysis.

    Table S5. Raw data.

    Movie S1. Time lapse showing Clec9a-tdT+ cell emergence.

    Movie S2. Time lapse displaying dead cell incorporation.

    References (5355)

  • Supplementary Materials

    The PDF file includes:

    • Methods
    • Fig. S1. Candidate transcription factors to instruct DC cell fate.
    • Fig. S2. Clec9a-based reporter to identify transcription factors to induce DC fate.
    • Fig. S3. Screening for transcription factors to activate Clec9a-tdT.
    • Fig. S4. DC morphology and dendrites are established in mouse fibroblasts.
    • Fig. S5. Spi1, Irf8, and Batf3 are enriched in cDC1 cells.
    • Fig. S6. Global single-cell gene expression analysis during iDC reprogramming.
    • Fig. S7. Stepwise transitions during iDC reprogramming.
    • Fig. S8. iDC reprogramming does not induce alterations associated with tumorigenesis.
    • Fig. S9. Analysis of iDC maturation states.
    • Fig. S10. Induced DCs are functional APCs.
    • Fig. S11. Polycistronic vector induces cDC1-like transcriptional program.
    • Fig. S12. PIB induce human iDC reprogramming.
    • Fig. S13. FACS gating strategies.
    • Legends for tables S1 to S5
    • Legends for movies S1 and S2
    • References (5355)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Analysis of candidate transcription factors.
    • Table S2 (Microsoft Excel format). Single-cell mRNA-seq data analysis.
    • Table S3 (Microsoft Excel format). Pseudotime single-cell mRNA-seq data analysis.
    • Table S4 (Microsoft Excel format). Population mRNA-seq data analysis.
    • Table S5 (Microsoft Excel format). Raw data.
    • Movie S1 (.avi format). Time lapse showing Clec9a-tdT+ cell emergence.
    • Movie S2 (.mp4 format). Time lapse displaying dead cell incorporation.

    Files in this Data Supplement:

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