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Batf3 maintains autoactivation of Irf8 for commitment of a CD8α+ conventional DC clonogenic progenitor

Nature Immunology volume 16, pages 708–717 (2015)Cite this article

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Abstract

The transcription factors Batf3 and IRF8 are required for the development of CD8α+ conventional dendritic cells (cDCs), but the basis for their actions has remained unclear. Here we identified two progenitor cells positive for the transcription factor Zbtb46 that separately generated CD8α+ cDCs and CD4+ cDCs and arose directly from the common DC progenitor (CDP). Irf8 expression in CDPs required prior autoactivation of Irf8 that was dependent on the transcription factor PU.1. Specification of the clonogenic progenitor of CD8α+ cDCs (the pre-CD8 DC) required IRF8 but not Batf3. However, after specification of pre-CD8 DCs, autoactivation of Irf8 became Batf3 dependent at a CD8α+ cDC–specific enhancer with multiple transcription factor AP1-IRF composite elements (AICEs) within the Irf8 superenhancer. CDPs from Batf3−/− mice that were specified toward development into pre-CD8 DCs failed to complete their development into CD8α+ cDCs due to decay of Irf8 autoactivation and diverted to the CD4+ cDC lineage.

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Figure 1: IRF8 is regulated by PU.1-dependent autoactivation of Irf8 in CDPs.
Figure 2: Batf3 is required for IRF8 expression in CD8α+ cDCs.
Figure 3: Induction of Zbtb46 identifies a clonogenic CD24+ cDC–committed progenitor in BM.
Figure 4: Specification of pre-CD8 DCs requires IRF8 but not Batf3.
Figure 5: Pre-CD8 DCs represent a development stage distinct from that of CDPs and mature CD24+ cDCs.
Figure 6: A Batf3-dependent Irf8 enhancer functions selectively in CD24+ cDCs.
Figure 7: Specified pre-CD8 DCs are diverted to the CD172a+ lineage in the absence of Batf3.
Figure 8: Transgenic overexpression of IRF8 bypasses the Batf3 requirement in CD8α+ cDC development.

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References

  1. Satpathy, A.T. et al. Re(de)fining the dendritic cell lineage. Nat. Immunol. 13, 1145–1154 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Cervantes-Barragan, L. et al. Plasmacytoid dendritic cells control T-cell response to chronic viral infection. Proc. Natl. Acad. Sci. USA 109, 3012–3017 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Reizis, B. et al. Plasmacytoid dendritic cells: recent progress and open questions. Annu. Rev. Immunol. 29, 163–183 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Vremec, D. et al. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J. Immunol. 164, 2978–2986 (2000).

    Article  CAS  PubMed  Google Scholar 

  5. Aliberti, J. et al. Essential role for ICSBP in the in vivo development of murine CD8α+ dendritic cells. Blood 101, 305–310 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Tamura, T. et al. IFN regulatory factor-4 and -8 govern dendritic cell subset development and their functional diversity. J. Immunol. 174, 2573–2581 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Hildner, K. et al. Batf3 deficiency reveals a critical role for CD8α+ dendritic cells in cytotoxic T cell immunity. Science 322, 1097–1100 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Torti, N. et al. Batf3 transcription factor-dependent DC subsets in murine CMV infection: differential impact on T-cell priming and memory inflation. Eur. J. Immunol. 41, 2612–2618 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Persson, E.K. et al. IRF4 Transcription-factor-dependent CD103+CD11b+ dendritic cells drive hucosal T helper 17 cell differentiation. Immunity 38, 958–969 (2013).

    Article  CAS  PubMed  Google Scholar 

  10. Gao, Y. et al. Control of T helper 2 responses by transcription factor IRF4-dependent dendritic cells. Immunity 39, 722–732 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Schlitzer, A. et al. IRF4 Transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses. Immunity 38, 970–983 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Merad, M. & Ginhoux, F. Dendritic cell genealogy: a new stem or just another branch? Nat. Immunol. 8, 1199–1201 (2007).

    Article  CAS  PubMed  Google Scholar 

  13. Onai, N. et al. Identification of clonogenic common Flt3+ M-CSFR+ plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow. Nat. Immunol. 8, 1207–1216 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Naik, S.H. et al. Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo. Nat. Immunol. 8, 1217–1226 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Schiavoni, G. et al. ICSBP is essential for the development of mouse type I interferon-producing cells and for the generation and activation of CD8α+ dendritic cells. J. Exp. Med. 196, 1415–1425 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Suzuki, S. et al. Critical roles of interferon regulatory factor 4 in CD11bhighCD8α− dendritic cell development. Proc. Natl. Acad. Sci. USA 101, 8981–8986 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Tailor, P. et al. The BXH2 mutation in IRF8 differentially impairs dendritic cell subset development in the mouse. Blood 111, 1942–1945 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kantakamalakul, W. et al. Regulation of IFN consensus sequence binding protein expression in murine macrophages. J. Immunol. 162, 7417–7425 (1999).

    CAS  PubMed  Google Scholar 

  19. Bornstein, C. et al. A negative feedback loop of transcription factors specifies alternative dendritic cell chromatin States. Mol. Cell 56, 749–762 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Vander, L.B. et al. Transcriptional programming of dendritic cells for enhanced MHC class II antigen presentation. Nat. Immunol. 15, 161–167 (2014).

    Article  CAS  Google Scholar 

  21. Hacker, C. et al. Transcriptional profiling identifies Id2 function in dendritic cell development. Nat. Immunol. 4, 380–386 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Spits, H. et al. Id2 and Id3 inhibit development of CD34+ stem cells into predendritic cell (pre-DC)2 but not into pre-DC1. Evidence for a lymphoid origin of pre-DC2. J. Exp. Med. 192, 1775–1784 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Li, H.S. et al. The signal transducers STAT5 and STAT3 control expression of Id2 and E2–2 during dendritic cell development. Blood 120, 4363–4373 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ghosh, H.S. et al. ETO family protein Mtg16 regulates the balance of dendritic cell subsets by repressing Id2. J. Exp. Med. 211, 1623–1635 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Tussiwand, R. et al. Compensatory dendritic cell development mediated by BATF-IRF interactions. Nature 490, 502–507 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Glasmacher, E. et al. A genomic regulatory element that directs assembly and function of immune-specific AP-1-IRF complexes. Science 338, 975–980 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Edelson, B.T. et al. Peripheral CD103+ dendritic cells form a unified subset developmentally related to CD8α+ conventional dendritic cells. J. Exp. Med. 207, 823–836 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Mashayekhi, M. et al. CD8α+ dendritic cells are the critical source of interleukin-12 that controls acute infection by Toxoplasma gondii tachyzoites. Immunity 35, 249–259 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Medvedovic, J. et al. Pax5: a master regulator of B cell development and leukemogenesis. Adv. Immunol. 111, 179–206 (2011).

    Article  CAS  PubMed  Google Scholar 

  30. Veraksa, A., Del Campo, M. & McGinnis, W. Developmental patterning genes and their conserved functions: from model organisms to humans. Mol. Genet. Metab. 69, 85–100 (2000).

    Article  CAS  PubMed  Google Scholar 

  31. Ouyang, W. et al. Stat6-independent GATA-3 autoactivation directs IL-4-independent Th2 development and commitment. Immunity 12, 27–37 (2000).

    Article  CAS  PubMed  Google Scholar 

  32. Boyer, L.A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Fogg, D.K. et al. A clonogenic bone marrow progenitor specific for macrophages and dendritic cells. Science 311, 83–87 (2006).

    Article  CAS  PubMed  Google Scholar 

  34. Wang, H. et al. A reporter mouse reveals lineage-specific and heterogeneous expression of IRF8 during lymphoid and myeloid cell differentiation. J. Immunol. 193, 1766–1777 (2014).

    Article  CAS  PubMed  Google Scholar 

  35. Liu, K. et al. In vivo analysis of dendritic cell development and homeostasis. Science 324, 392–397 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Onai, N. et al. A clonogenic progenitor with prominent plasmacytoid dendritic cell developmental potential. Immunity 38, 943–957 (2013).

    Article  CAS  PubMed  Google Scholar 

  37. Satpathy, A.T. et al. Zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages. J. Exp. Med. 209, 1135–1152 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Naik, S.H. et al. Intrasplenic steady-state dendritic cell precursors that are distinct from monocytes. Nat. Immunol. 7, 663–671 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Whyte, W.A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013).

    Article  CAS  PubMed  Google Scholar 

  41. Zhu, H. et al. Unexpected characteristics of the IFN-γ reporters in nontransformed T cells. J. Immunol. 167, 855–865 (2001).

    Article  CAS  PubMed  Google Scholar 

  42. Schraml, B.U. et al. The AP-1 transcription factor Batf controls TH17 differentiation. Nature 460, 405–409 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Ise, W. et al. The transcription factor BATF controls the global regulators of class-switch recombination in both B cells and T cells. Nat. Immunol. 12, 536–543 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Schönheit, J. et al. PU.1 level-directed chromatin structure remodeling at the Irf8 gene drives dendritic cell commitment. Cell Rep. 3, 1617–1628 (2013).

    Article  CAS  PubMed  Google Scholar 

  45. Reizis, B. et al. Plasmacytoid dendritic cells: one-trick ponies or workhorses of the immune system? Nat. Rev. Immunol. 11, 558–565 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Ichikawa, E. et al. Defective development of splenic and epidermal CD4+ dendritic cells in mice deficient for IFN regulatory factor-2. Proc. Natl. Acad. Sci. USA 101, 3909–3914 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Nagasawa, M. et al. Development of human plasmacytoid dendritic cells depends on the combined action of the basic helix-loop-helix factor E2–2 and the Ets factor Spi-B. Eur. J. Immunol. 38, 2389–2400 (2008).

    Article  CAS  PubMed  Google Scholar 

  48. Xiong, H. et al. Ubiquitin-dependent degradation of interferon regulatory factor-8 mediated by Cbl down-regulates interleukin-12 expression. J. Biol. Chem. 280, 23531–23539 (2005).

    Article  CAS  PubMed  Google Scholar 

  49. Edelson, B.T. et al. CD8α+ dendritic cells are an obligate cellular entry point for productive infection by Listeria monocytogenes. Immunity 35, 236–248 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Naik, S.H. et al. Cutting edge: generation of splenic CD8+ and CD8– dendritic cell equivalents in Fms-like tyrosine kinase 3 ligand bone marrow cultures. J. Immunol. 174, 6592–6597 (2005).

    Article  CAS  PubMed  Google Scholar 

  51. Kanbe, E. & Zhang, D.E. A simple and quick method to concentrate MSCV retrovirus. Blood Cells Mol. Dis. 33, 64–67 (2004).

    Article  CAS  PubMed  Google Scholar 

  52. Shooshtari, P. et al. Correlation analysis of intracellular and secreted cytokines via the generalized integrated mean fluorescence intensity. Cytometry A 77, 873–880 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Darrah, P.A. et al. Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nat. Med. 13, 843–850 (2007).

    Article  CAS  PubMed  Google Scholar 

  54. KC, W, et al. L-Myc expression by dendritic cells is required for optimal T-cell priming. Nature 507, 243–247 (2014).

    Article  CAS  Google Scholar 

  55. Langmead, B. et al. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    PubMed  PubMed Central  Google Scholar 

  56. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    PubMed  PubMed Central  Google Scholar 

  57. ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

  58. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Bailey, T.L. et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Lovén, J. et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 153, 320–334 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the Alvin J. Siteman Cancer Center at Washington University School of Medicine for use of the Center for Biomedical Informatics and Multiplex Gene Analysis Genechip Core Facility. Supported by the Howard Hughes Medical Institute, the US National Institutes of Health (1F31CA189491-01 to G.E.G.-R. and K08AI106953 to M.H.), the American Heart Association (12PRE12050419 to W.K.), and the National Cancer Institute (P30 CA91842; for support of the Alvin J. Siteman Cancer Center).

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Authors and Affiliations

  1. Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA

    Gary E Grajales-Reyes, Arifumi Iwata, Xiaodi Wu, Roxane Tussiwand, Wumesh KC, Nicole M Kretzer, Carlos G Briseño, Vivek Durai, Prachi Bagadia, Malay Haldar, Theresa L Murphy & Kenneth M Murphy

  2. Department of Medicine A, Hematology and Oncology, University of Muenster, Muenster, Germany

    Jörn Albring

  3. Department of Biomedicine, University of Basel, Basel, Switzerland

    Roxane Tussiwand

  4. Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany

    Jörg Schönheit

  5. Institute of Molecular Tumor Biology, University of Münster, Münster, Germany

    Frank Rosenbauer

  6. Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri, USA

    Kenneth M Murphy

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Contributions

G.E.G.-R., T.L.M. and K.M.M. designed the study; G.E.G.-R., A.I., J.A., R.T. and T.L.M. performed experiments related to cell sorting, retroviral infection, culture and flow cytometry, with advice from N.M.K., C.G.B., V.D., P.B. and M.H.; W.K. performed experiments related to antibody generation and Irf8 ChIP-Seq; A.I. performed and analyzed ChIP-Seq experiments; G.E.G.-R. performed microarray experiments with advice from and analysis by X.W. and C.G.B.; J.S. and F.R. provided Irf8VENUS mice and advice; and G.E.G.-R., X.W., T.L.M. and K.M.M. wrote the manuscript with contributions from all authors.

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Integrated supplementary information

Supplementary Figure 1 CD115 heterogeneity in the pre-cDC identifies populations with different DC lineage potential.

(a) Splenocytes from the indicated genotype were analyzed for CD8α+ and CD205+ expression on cDCs (CD11c+ MHCII+ B220-). Data is representative of 2 mice from each genotype. (b) BM from WT mice was analyzed for expression of CD115 in pre-cDCs, defined as Lin− Flt3+ MHClow CD11c+ BM cells (n=5). Lin includes MHC II, CD19, B220, CD3, NK1.1, CD105, TER-119. (c) CDPs, sorted as Lin- CD117int-low CD135+ CD115+ CD11c- BM cells, or pre-cDCs purified as CD115+ or CD115- fractions, as indicated, were cultured with FLT3L for 4 days, and analyzed by FACS for development of CD24+ cDC, CD172a+ cDCs (upper panels), or pDCs (lower panels). (n=5). (d) Shown is the average percentage of CD24+ cDCs or pDCs that develop from the fractions in (c), one-way ANOVA, Tukey’s post-hoc test, error bars show s.e.m. Numbers represent the percent of cells in the indicated gates. (***P < 0.001).

Supplementary Figure 2 CD117int Zbtb46GFP+ pre-CD8 DCs develop directly from the CDP.

(a) Shown is the gating strategy to sort purify CD117int CDP from WT BM cells. Lin includes CD19, B220, CD3, NK1.1, CD105, TER-119. (b) Time course of CD117int CDPs in FLT3L-treated cultures analyzed by FACS after 1, 2 and 3 days as indicated, for the development of CD117int Zbtb46GFP+ pre-CD8 cDCs (n=3). CD117 expression is shown against Zbtb46GFP (upper panels), MHC II (middle panels), and CD11c (lower panels), respectively. Numbers are the percent of cells in the indicated gates.

Supplementary Figure 3 BATF3, IRF8 and p300 bind a cis element in the Irf8 locus +32 kb from the Irf8 TSS.

(a) Shown is the expression value for Irf8 in pDCs, CD8α+ DCs and CD4 DCs using values obtained from the Immgen database. (b) Shown is the expression value for Batf3 in pDCs, CD8α+ DCs and CD4 DCs using values obtained from the Immgen database. (c) Shown are high resolution views of ChIP-seq data for the indicated antigens in pDCs, CD172a+ cDCs and CD24+ cDCs. Genomic regions that contain IRF8 peaks in CD24+ cDCs located -26kb, -16kb, +32kb and +41kb relative to the Irf8 TSS are expanded.

Supplementary Figure 4 The Irf8 locus contains a superenhancer in both pDCs and CD8α+ cDCs.

(a-c) Distribution of enhancers identified using the H3K27ac ChIP-seq signal in CD24+ cDCs (a), CD172a+ cDCs (b) and pDCs (c). (d) ChIP-seq data for the indicated target in CD24+ cDCs, CD172a+ cDCs and pDCs for the Id2, Itgae and Bcl11a loci. Regions ranked as superenhancers using normalized H3K27ac signal are indicated by gray bars.

Supplementary Figure 5 The cis element +32 kb from the Irf8 TSS is specifically active in CD8α+ cDCs.

(a) Shown is the structure of the retroviral reporter construct. hCD4 expression indicates positive retroviral infection and GFP expression indicates activity of enhancer elements cloned upstream of a minimal Irf8 promoter. (b) Activity of enhancer elements in CD172+ cDCs from WT (black triangles) and Batf3-/- (open triangles) mice is shown as integrated MFI. (c) WT and Batf3-/- BM cells enriched using anti-CD117 magnetic beads (Miltenyi Biotec) were cultured in FLT3L and infected with retroviral reporters containing enhancer elements upstream of a minimal Irf8 promoter. On day 7, infected cDCs were identified as hCD4+ and analyzed for GFP expression in CD172a+ and CD172a- populations gated as indicated. (d) hCD4+ pDCs, gated as indicated, were analyzed for GFP expression.

Supplementary Figure 6 The cis element +32 kb from the Irf8 TSS contains four AICE motifs.

Shown is the AICE motif found by MEME analysis in 598 out of the top 1000 Batf3 ChIP-seq peaks in CD24+ cDCs. (b) Shown are 4 sites in the +32kb Irf8 enhancer element identified by FIMO using the AICE PWM from (a). (c) Shown are the locations and mammalian conservation of AICE sites 1-4 in relation to the Batf3 and IRF8 ChIP-seq peaks in CD24+ cDCs within the +32kb Irf8 enhancer. Black bars indicate 2 regions of high mammalian conservation. (d) Shown is the ClustalW alignment between sequences of mouse and human for conserved region 1 from the +32kb Irf8 enhancer with sites 1, 4 and 3 boxed. (e) Shown is the ClustalW alignment between sequences of mouse and human for conserved region 2 from the +32kb Irf8 enhancer with site 2 boxed.

Supplementary Figure 7 The committed pre-CD4 DC progenitor initially expresses IRF8 but rapidly extinguishes its expression.

(a) WT BM cells were analyzed for IRF8 expression in pre-CD4 DCs. Lin- FLT3+ CD115+ CD117- CD11c+ cells (left panel) are analyzed for intracellular IRF8 and CD24 expression (right panel). Numbers are the percent of cells in the indicated gates. (b) CDPs and pre-CD4 DCs in (a) were purified by cell sorting and cultured with FLT3L for 1 and 3 days, and analyzed for intracellular IRF8 expression by FACS. Shown are the percentages of cells in the indicated gates.

Supplementary Figure 8 VENUS expression in Irf8VENUS+ mice reflects the pattern expected for IRF8 in various DC subsets.

(a) Splenocytes from WT and Irf8VENUS+ mice were analyzed by FACS for the indicated markers. Upper panels show the gating strategy to identify CD24+ cDCs, CD172a+ cDCs and pDCs. pDCs are identified as CD317+ B220+ live cells (left panels). B220- MHCII+ CD11c+ cells were gated as either CD24-CD172a+ or CD24+ CD172a- populations. (b) DC subsets from WT (shaded) or Irf8-VENUS+ (black line) mice that were pre-gated in (a) were analyzed in a single parameter histogram for expression of YFP VENUS. Numbers represent the percentage of cells from Irf8VENUS mice in the indicated gates.

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Grajales-Reyes, G., Iwata, A., Albring, J. et al. Batf3 maintains autoactivation of Irf8 for commitment of a CD8α+ conventional DC clonogenic progenitor. Nat Immunol 16, 708–717 (2015). https://doi.org/10.1038/ni.3197

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