Abstract
Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. In the present study Irf8 enhancers were identified via chromatin profiling of dendritic cells and CRISPR/Cas9 genome editing was used to assess their roles in Irf8 regulation. An enhancer 32 kilobases (kb) downstream of the Irf8 transcriptional start site (+32-kb Irf8) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41-kb Irf8 enhancer, previously thought to be active only in plasmacytoid dendritic cells, was found to also be transiently accessible in cDC1 progenitors, and deleting this enhancer prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41-kb Irf8 enhancer in dendritic cell progenitors is responsible for cDC1 fate specification.
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Data availability.
The sequencing and microarray data generated during the course of this study have been deposited and are available on the Gene Expression Omnibus database. The ChIP-seq data of DC progenitors used in Fig. 3 can be accessed with the following accession number: GSE132239. The ATAC-seq data of DC progenitors used in Figs. 5 and 6 can be accessed with the following accession number: GSE132240. The microarrays utilized in Supplementary Fig. 4 can be accessed with the following accession numbers: GSE123747, GSE132767 and GSE132768.
All other primary data and materials that support the findings of this study are available from the corresponding author upon request.
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Acknowledgements
This work was supported by the Howard Hughes Medical Institute (K.M. and H.C.), the US National Institutes of Health (NIH: F30 DK108498 to V.D.; K08 CA230188 to A.S.; P50 HG007735 to H.C.; R01 AI106352 to B.K.; R01 DK097317 to R.N.), the National Science Foundation (DGE-1745038 to P.B.), the Parker Institute for Cancer Immunotherapy (A.S. and H.C.) and Boehringer Ingelheim (M.W., H.T. and M.B.). A.S. was supported by a Career Award for Medical Scientists from the Burroughs Wellcome Fund. This work benefitted from data assembled by the ImmGen consortium48. We thank the Genome Technology Access Center in the Department of Genetics at Washington University in St Louis School of Medicine for help with genomic analysis. The Center is partially supported by the National Cancer Institute’s Cancer Center Support grant no. P30 CA91842 to the Siteman Cancer Center and by the Institute of Clinical and Translational Sciences/Clinical and Translational Science Award grant no. UL1TR000448 from the National Center for Research Resources (NCRR), a component of the NIH, and NIH Roadmap for Medical Research. This publication is solely the responsibility of the authors and does not necessarily represent the official view of the NCRR or the NIH.
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V.D., T.L.M. and K.M.M. designed the study. V.D. generated the enhancer knockout mice with advice and reagents from S.J.P. V.D. and P.B. performed experiments related to analysis of immune populations, cell sorting and culture, with advice from J.T.D., R.W., T.-T.L., X.H., C.G.B. and G.E.G.-R. V.D., J.M.G., A.T.S. and H.Y.C. performed ATAC-seq of DC progenitors. J.M.G., A.T.S. and H.Y.C. performed computational analysis of ATAC-seq data. D.H.K. and R.D.N. performed Salmonella infections. A.I. performed ChIP-seq and computational analysis on DC progenitors. M.W., H.T. and M.B. provided ChIP-seq and ATAC-seq data from B cells and advice. B.L.K. provided Tcf3–/– mice and advice. V.D. and K.M.M. wrote the manuscript with advice from all the authors.
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Supplementary Figure 1 AICEs in the +32-kb Irf8 enhancer drive cDC1 development.
a, Shown are the sequences, either wild type (3x AICE1 WT) or mutated (3x AICE1 Mut), of the first 3 AICEs from the +32 kb Irf8 enhancer used in retroviral reporter assays in (b). b, The regions from (a) were placed in a reverse strand retroviral reporter and GFP activity was measured in either cDC1s or cDC2s as previously described21. Gray histograms represent the activity of the empty reporter. Data are representative of three independent experiments with similar results (n = 3 biological replicates per group). c, The full 574 bp +32 kb Irf8 enhancer (+32kb WT), or the 5’ half containing 4 AICE sites (+32 kb 5’) or the 3’ half containing no AICEs (+32 kb 3’), were assayed for activity in cDC1s using retroviral reporters as in (b). Numbers indicate the mean fluorescence intensity (MFI) of GFP in cDC1s. Data are representative of three independent experiments with similar results (n = 3 biological replicates per group). d, CD117hi BM cells from R26Cas9/+ mice were infected with a retroviral vector expressing a control scramble sgRNA (scramble), an sgRNA targeting the Irf8 coding sequence (αIrf8), or an sgRNA targeting the AICE site 4 shown in (a) (αIrf8 +32kb), and then cultured in Flt3L for 8 days. Flow cytometry was then used to assess cDC1 development. Cells are pre-gated as Thy1.1+, to identify infected cells, and as B220—MHC-II+CD11c+, to identify DCs. Numbers indicate the percent of cells in the indicated gates. Data are representative of three independent experiments (n = 3 biological replicates per group). e, Shown is the nucleotide sequence of the +32 kb Irf8 enhancer region. The extent of deletion in the two mouse strains is indicated by colored fonts. Pink nucleotides were deleted only in Irf8 +32 5’−/− mice, blue nucleotides were deleted in both Irf8 +32 5’−/− mice and Irf8 +32−/− mice, and red nucleotides were deleted only in Irf8 +32–/– mice. Red lines indicate the sequences of the sgRNAs used for CRISPR/Cas9 induced deletion in mice. Green boxes indicate the AICE motifs. f, PCR of the +32 kb locus in wildtype (WT), Irf8 +32 5’−/−, and Irf8 +32−/− mice demonstrating the deletions produced by CRISPR/Cas9 genome editing. Numbers indicate the size of the PCR product from each strain. Data are representative of two independent experiments with similar results (n = 2 biological replicates per population). g,h, Flow cytometry of live splenocytes from mice of the indicated genotypes was used to identify dendritic cell (DC) subsets. Numbers indicate the percent of cells in the indicated gates (g). Statistical analysis of the frequency and absolute number of splenic cDC1s in mice of the indicated genotypes (h). Small horizontal lines indicate the mean. Data are pooled from three independent experiments (n = 6 mice for WT, n = 5 mice for Irf8 +32 5’–/–, and n = 5 mice for Irf8 +32–/–). ***P<0.001; and ****P<0.0001, unpaired two-tailed Student’s t-test (h).
Supplementary Figure 2 Loss of the +32-kb Irf8 enhancer does not impact non-cDC1 immune lineages.
a,b, Flow cytometry of live splenocytes from mice of the indicated genotypes was used to examine myeloid and lymphoid cells. Numbers indicate the percent of cells in the indicated gates (a). Statistical analysis of the frequency of the indicated populations within spleens of mice of the indicated genotypes (b). Small horizontal lines indicate the mean. Data are pooled from four independent experiments (n = 5 mice for WT, n = 5 mice for Irf8 +32–/–, and n = 3 mice for Irf8–/–). c, Intracellular staining for IRF8 in the indicated populations in mice of the indicated genotypes. Data are representative of four independent experiments with similar results (n = 5 mice for WT, n = 5 mice for Irf8 +32–/–, and n = 3 mice for Irf8–/–). ns, not significant (P>0.05); **P<0.01; ***P<0.001; and ****P<0.0001, ordinary one-way ANOVA (b).
Supplementary Figure 3 The +32-kb Irf8 enhancer is required for all compensatory cDC1 development.
a,b, Flow cytometry of lung cells from mice of the indicated genotypes was used to identify DC subsets. Numbers indicate the percent of cells in the indicated gates (a). Statistical analysis of the frequency of cDC1s among CD45+ cells from the lungs of mice of the indicated genotypes (b). Small horizontal lines indicate the mean. Data are pooled from four independent experiments (n = 5 mice for WT, n = 5 mice for Irf8 +32–/–, n = 3 mice for Batf3–/–, and n = 3 mice for Irf8–/–). c,d, BM cells from mice of the indicated genotypes was transferred into irradiated WT mice and allowed to reconstitute for three weeks. Flow cytometry of live splenocytes from these chimeras was then used to identify DC subsets. Numbers indicate the percent of cells in the indicated gates (c). Statistical analysis of the frequency of splenic cDC1s in the indicated chimeras (d). Small horizontal lines indicate the mean. Data are pooled from two independent experiments (n = 3 for WT into WT chimeras, n = 5 for Irf8 +32–/– into WT chimeras, n = 5 for Batf3–/– into WT chimeras). e, Statistical analysis of the frequency of pre-cDC1s in mice of the indicated genotypes. Small horizontal lines indicate the mean. Data are pooled from three independent experiments (n = 3 mice for WT and n = 3 mice for Irf8 +32–/–). ns, not significant (P>0.05); **P<0.01, ordinary one-way ANOVA (b,d) or unpaired two-tailed Student’s t-test (e).
Supplementary Figure 4 CD226 uniquely identifies pre-cDC1s in the bone marrow.
a, Cd226 gene expression levels in CDPs and pre-cDC1s quantified by gene expression microarrays. Small horizontal lines indicate the mean. Data are pooled from three independent experiments (n = 3 biological replicates per population). b, Flow cytometry of Lin— BM cells from WT mice was used to examine marker expression in pre-cDC1s. Data are representative of four independent experiments with similar results (n = 4 mice). c,d, CDPs (Lin—CD117intCD135+CD115+) and Zbtb46-GFP+ pre-cDC1s (Lin—CD117intCD135+Zbtb46-GFP+) (c), or CDPs and CD226+ pre-cDC1s (Lin—CD117intCD135+CD226+) (d) were sorted from mice and cultured for 5 days in Flt3L. Cells were then analyzed by flow cytometry to identify dendritic cell subsets. Data are representative of three independent experiments with similar results (n = 3 biological replicates per population). e, Microarray analysis of cDC2s derived from in vitro culture of pre-cDC1s from Irf8 +32–/– mice compared to cDC1s derived from in vitro culture of WT pre-cDC1s (left) or compared to cDC2s derived from in vitro culture of WT pre-cDC2s (right), presented as M-plots. Green lines indicate the threshold for a two-fold difference in expression between samples. Data was pooled from two independent experiments (n = 2 biological replicates per population). f, Microarray analysis of WT CD24+CD172a+ cDC2s compared to WT CD24–CD172+ cDC2s (left) or compared to WT CD24+CD172a– cDC1s (right), presented as M-plots. Green lines indicate the threshold for a two-fold difference in expression between samples. Data was pooled from two independent experiments (n = 2 biological replicates per population). g, Irf4 expression levels in the indicated populations quantified by gene expression microarrays. Small horizontal lines indicate the mean. Data was pooled from two independent experiments (n = 2 biological replicates per population). h, Microarray analysis of cDC2s derived from in vitro culture of Batf3–/– pre-cDC1s compared to cDC1s derived from in vitro culture of WT pre-cDC1s (left) or compared to cDC2s derived from in vitro culture of WT pre-cDC2s (right), presented as M-plots. Green lines indicate the threshold for a two-fold difference in expression between samples. Data was pooled from two independent experiments (n = 2 biological replicates per population).
Supplementary Figure 5 Loss of the −50-kb Irf8 enhancer does not impact non-monocyte/macrophage immune lineages.
a, PCR of the -50 kb locus in WT and Irf8 -50–/– mice demonstrating the deletion produced by CRISPR/Cas9 genome editing. Numbers indicate the size of the PCR product from each strain. Data are representative of two independent experiments with similar results (n = 2 biological replicates per population). b, Shown is the nucleotide sequence of the -50 kb Irf8 enhancer region. The extent of deletion in Irf8 -50—/— mice is indicated by colored font. Red nucleotides were deleted in Irf8 -50—/— mice. Red lines indicate the sequences of the sgRNAs used for CRISPR/Cas9 induced deletion in mice. Purple boxes indicate the PU.1 motifs. c, Statistical analysis of the frequency of the indicated populations gated as in Supplementary Fig. 2a in spleens of mice of the indicated genotypes. Small horizontal lines indicate the mean. Data are pooled from four independent experiments. (n = 5 mice for WT, n = 5 mice for Irf8 -50–/–, and n = 4 mice for Irf8–/–). d, Intracellular staining for IRF8 in the indicated populations in mice of the indicated genotypes. Data are representative of four independent experiments with similar results (n = 5 mice for WT, n = 5 mice for Irf8 -50–/–, and n = 4 mice for Irf8–/–). ns, not significant (P>0.05); *P<0.05; **P<0.01; and ****P<0.0001, ordinary one-way ANOVA (c).
Supplementary Figure 6 Loss of the +41-kb Irf8 enhancer does not impact non-DC immune lineages.
a, PCR of the +41 kb locus in WT and Irf8 +41–/– mice demonstrating the deletion produced by CRISPR/Cas9 genome editing. Numbers indicate the size of the PCR product from each strain. Data are representative of two independent experiments with similar results (n = 2 biological replicates per population). b, Shown is the nucleotide sequence of the +41-kb Irf8 enhancer region. The extent of deletion in Irf8 +41–/– mice is indicated by colored font. Red nucleotides were deleted in Irf8 +41–/– mice. Red lines indicate the sequences of the sgRNAs used for CRISPR/Cas9 induced deletion in mice. Blue boxes indicate the E box motifs. c, Statistical analysis of the frequency of the indicated populations gated as in Supplementary Fig. 2a in spleens of mice of the indicated genotypes. Small horizontal lines indicate the mean. Data are pooled from five independent experiments (n = 7 mice for WT, n = 7 mice for Irf8 +41–/–, and n = 4 mice for Irf8–/–). d, Intracellular staining for IRF8 in the indicated populations in mice of the indicated genotypes. Data are representative of four independent experiments with similar results (n = 5 mice for WT, n = 5 mice for Irf8 +41–/–, and n = 3 mice for Irf8–/–). ns, not significant (P>0.05); ***P<0.001; and ****P<0.0001, ordinary one-way ANOVA (c).
Supplementary Figure 7 Tcf3–/– BM progenitors have reduced cDC1 and pDC potential.
a, Flow cytometry of BM cells from mice of the indicated genotypes was used to determine intracellular E2A-GFP levels in the indicated populations. CMPs were gated as Lin–CD117hiSca-1–CD16/32+CD34+, CLPs were gated as Lin–CD127+CD135+CD117loB220–CD11c–, and pre-B cells were gated as Lin–CD19+CD25+. Numbers indicate the MFI of E2A-GFP levels. Data are representative of two independent experiments with similar results (n = 3 mice for Tcf3fl/+ and n = 3 mice for Tcf3fl/+VavCretg). b,c, Flow cytometry of live splenocytes from mice of the indicated genotypes was used to identify DC subsets. Numbers indicate the percent of cells in the indicated gates (b). Statistical analysis of the frequency of splenic cDC1s and pDCs in mice of the indicated genotypes (c). Small horizontal lines indicate the mean. Data are pooled from three independent experiments. (n = 4 mice for WT and n = 3 mice for Tcf3–/–). d,e, WT CD45.1 mice were lethally irradiated and reconstituted with Tcf3+/+ CD45.2: Tcf3+/+ CD45.1 BM at a 1:1 ratio or Tcf3+/+ CD45.2: Tcf3–/– CD45.1 BM at a 1:1 ratio. Mice were analyzed after four weeks of reconstitution. Flow cytometry of live splenocytes from the indicated chimeras was then used to identify DC subsets gated as the following: cDC2s, CD11c+MHCII+CD172a+; cDC1s, CD11c+MHCII+CD24+CD172a–; pDCs, B220+CD317+. Numbers indicate the percent of cells in the indicated gates (d). Summary data of the chimerism ratio of splenic cDC1s (left) or pDCs (right) presented as the ratio of CD45.1+ to CD45.2+ cells of each type normalized to the ratio of CD45.1+ to CD45.2+ cDC2s within the same mouse (e). Small horizontal lines indicate the mean. Data are pooled from two independent experiments (n = 6 mice for Tcf3+/+:Tcf3+/+ chimeras and n = 4 mice for Tcf3+/+:Tcf3–/– chimeras). ns, not significant (P>0.05); *P<0.01; and ****P<0.0001, unpaired two-tailed Student’s t-test (c,e).
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Durai, V., Bagadia, P., Granja, J.M. et al. Cryptic activation of an Irf8 enhancer governs cDC1 fate specification. Nat Immunol 20, 1161–1173 (2019). https://doi.org/10.1038/s41590-019-0450-x
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DOI: https://doi.org/10.1038/s41590-019-0450-x
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