Abstract
Autoactivation of lineage-determining transcription factors mediates bistable expression, generating distinct cell phenotypes essential for complex body plans. Classical type 1 dendritic cell (cDC1) and type 2 dendritic cell (cDC2) subsets provide nonredundant functions for defense against distinct immune challenges. Interferon regulatory factor 8 (IRF8), the cDC1 lineage-determining transcription factor, undergoes autoactivation in cDC1 progenitors to establish cDC1 identity, yet its expression is downregulated during cDC2 differentiation by an unknown mechanism. This study reveals that the Irf8 +32-kb enhancer, responsible for IRF8 autoactivation, is naturally suboptimized with low-affinity IRF8 binding sites. Introducing multiple high-affinity IRF8 sites into the Irf8 +32-kb enhancer causes a gain-of-function effect, leading to erroneous IRF8 autoactivation in specified cDC2 progenitors, redirecting them toward cDC1 and a novel hybrid DC subset with mixed-lineage phenotypes. Further, this also causes a loss-of-function effect, reducing Irf8 expression in cDC1s. These developmental alterations critically impair both cDC1-dependent and cDC2-dependent arms of immunity. Collectively, our findings underscore the significance of enhancer suboptimization in the developmental segregation of cDCs required for normal immune function.
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Data availability
The bulk RNA-seq data generated in this study on splenic DC populations are available in the Gene Expression Omnibus under accession number GSE241341. The scRNA-seq data generated in this study are available under accession number GSE270060. The following datasets were previously published and reanalyzed in this study: microarrays on DC progenitors and splenic DCs (GSE66565)22, and ChIP–seq for BATF3 and IRF8 in cDC1 (GSE66899)22. The following datasets were reanalyzed previously19 and used in the current study: ChIP–seq for p300, H3K4me1 and H3K27ac in DC progenitors and DCs (GSE66899)22, the assay for transposase-accessible chromatin with sequencing (ATAC-seq) of DC progenitors (GSE132240)23, and Immunological Genome Project (ImmGen) ATAC-seq on splenic DCs (GSE100738)68. Mouse genome (mm10 assembly) Bowtie 2 indexes were downloaded from the Bowtie 2 website (https://genome-idx.s3.amazonaws.com/bt/mm10.zip). Source data are provided with this paper. All other data are available in the paper or Supplementary Information.
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Acknowledgements
This work was supported by grants from the US National Institute of Health (NIH) to K.M.M. (R01AI150297, R01CA248919, R21AI164142, R01AI162643 and R21AI163421). S.T.F. is a Cancer Research Institute Irvington Fellow supported by the Cancer Research Institute. We thank J. M. White at the Department of Pathology and Immunology Transgenic Mouse Core at Washington University in St. Louis and the Genetic Editing and iPS Cell Center at Washington University in St. Louis for generating the mouse models. We thank the GTAC@MGI at Washington University School of Medicine for sequencing services. We are grateful to C. Fan and C. A. Miller at Washington University School of Medicine, and Y. Zhou at Stanford University School of Medicine for their valuable advice on genomic analysis and S. Hui at Washington University School of Medicine for help with structural modeling. We acknowledge the NIH Tetramer Core Facility (contract no. 75N93020D00005) for providing SIINFEKL-Kb tetramers. We thank E. K. Farley at the University of California, San Diego, D. L. Stern at HHMI’s Janelia Research Campus and E. V. Rothenberg at the California Institute of Technology for insights about enhancer affinity and syntax.
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F.O., T.L.M. and K.M.M. designed the study. F.O., T.-T.L., P.D., S.T.F., S.K., H.S., R.A.O., S.J. and J.C. performed experiments with advice from J.L.P., S.D. and M.S.D. F.O. and K.M.M. wrote the manuscript with advice from T.L.M. and T.-T.L.
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M.S.D. is a consultant or advisor for Inbios, Vir Biotechnology, Moderna, Merck, GlaxoSmithKline, IntegerBio and Akagera Medicines. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Moderna, Vir Biotechnology, IntegerBio and Emergent BioSolutions. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 The Irf8 + 32 kb comprises four AICE motifs.
(a) ATAC-seq and ChIP-seq tracks for the indicated cell types are shown for the Irf8 locus. The locations of the Irf8 + 32 kb, +41 kb and +56 kb enhancers are highlighted. The tracks are displayed using the UCSC genome browser with vertical auto scale. (b) A zoomed-in view of a 1,168 bp region surrounding the Irf8 + 32 kb enhancer highlighting the locations of AICEs. The previously deleted 149 bp (blue) and 421 bp (red) regions from Irf8 + 32 5′−/− mice and Irf8 + 32−/− mice, respectively23, are indicated relative to the AICEs. (c) Sequence of the 421 bp region deleted previously in Irf8 + 32−/− mice23, with AICEs underlined and highlighted in blue. The AICEs are displayed from left to right in the order of site 1, site 4, site 3, and site 2.
Extended Data Fig. 2 AICEs in mouse and human IRF8 enhancers show low binding affinities to the BATF3-JUNB-IRF8 complexes.
(a) Competitive EMSA showing binding of 32P-dCTP-labeled Ctla4 probe with the indicated unlabeled competitors at increasing amounts (0, 25, 100, 400, and 1600−fold excess). The NEs used were combined from 293FT cells transfected with Junb and Batf3 and 293FT cells transfected with Irf8. Bands corresponding JUNB-BATF3-IRF8 complexes and free probes are indicated by the upper and lower arrows, respectively. Data shown are representative of two similar experiments. (b) EMSA performed with 32P-dCTP-labeled probes on various NEs: NEs from 293FT cells transfected with Junb and Batf3 (B), combined NEs from 293FT cells transfected with Junb and Batf3 and 293FT cells transfected with IRF8 (B + I), and NEs from MutuDCs. Bands corresponding JUNB-BATF3-IRF8 and JUNB-BATF3 complexes are indicated by the upper and lower arrows, respectively. Data shown are representative of three similar experiments. (c) Alignment of a 161 bp region encompassing the four AICEs in the mouse Irf8 + 32 kb enhancer (chr8:120,768,539-120,768,699, mm10) across several mammalian species, colored by conservation (Jalview). The six most probable AICEs (defined using FIMO and the AICE PWM from Fig. 1b) are indicated. (d) Retroviral reporter analysis of the mouse Irf8 + 32 kb enhancer and the human IRF8 + 48 kb enhancer in cDC1s and cDC2s obtained from KitL/Flt3L cultured mouse BM cells. Shown are enhancer-driven GFP expression in transduced cDC1s (pre-gate: B220- CD11c+ MHCII+ CD24+ Sirpa− Thy1.1+ cells) and transduced cDC2s (pre-gate: B220− CD11c+ MHCII+ Sirpa+ Thy1.1+ cells). Numbers are GFP geometric MFI. Data shown are representative of two similar experiments. (e) Competitive EMSA performed with 32P-dCTP-labeled Ctla4 probe in the presence of 200-fold excess of various unlabeled competitors. Competitors derived from the human +48 kb enhancer are denoted as ‘Hu’. Bands corresponding JUNB-BATF3-IRF8 complexes and free probes are indicated by the upper and lower arrows, respectively. Data shown are representative of two similar experiments.
Extended Data Fig. 3 Characterization of DC progenitors and splenic DC populations.
(a) Expression levels of selected TF-encoding genes in different stages of DC development assessed by microarray. (b) IRF8 Expression across different stages of DC development in WT mice as determined by intracellular staining. Numbers are geometric MFI. Data shown are representative of two similar experiments. MDP, monocyte-dendritic cell progenitors. (c) Frequencies of the indicated DC progenitors from WT, H/+, and H/H mice as percentages of lin− SiglecH− BM cells. Data are pooled from three independent experiments (n = 5 for WT, 3 for H/+, and 4 for H/H mice). Statistical significance was determined by one-way ANOVA. (d) Frequencies of splenic pDCs (gated as B220− SiglecH+ splenocytes) from WT, H/+, and H/H mice. Data are pooled from three independent experiments (n = 9 for WT, 5 for H/+, and 7 for H/H mice). Statistical significance was determined by one-way ANOVA. (e) ZBTB46 expression in the indicated cell types from H/H spleens measured by intracellular staining. B cells serve as a negative control for ZBTB46 expression. (f) In vitro cross-presentation assay with different DC populations. Shown are representative flow cytometry plots depicting CellTrace Violet-labeled OT-I CD8 T cells co-cultured with the indicated DC subsets and HKLM-OVA antigen for three days (pre-gate: CD45.2− CD45.1+ CD8a+ CD4− Va2+ cells). Divided OT-I cells indicate successful cross-presentation and T cell activation. (g) Frequencies of divided OT-I cells from in vitro cross-presentation assays. Data are pooled from two independent experiments. (h) Overlaid flow cytometry plots showing splenic cDC1s (cyan), cDC2s (red), and hybrid DCs (purple) before (top panel) and after (middle and bottom panels) culturing in the specified conditions. The data presented are representative of three similar experiments. Data in c and d are presented as mean values +/− SD.
Extended Data Fig. 4 An optimized Irf8 + 32 kb enhancer maintains IRF8 expression in pre-cDC2, impeding their lineage commitment to cDC2.
(a) IRF8 Expression in the indicated DC progenitors from WT and Irf8 + 32H/H (H/H) mice as determined by intracellular staining. Numbers are geometric MFI. Data shown are representative of two similar experiments. (b) Representative flow cytometry plots depicting intracellular IRF8 in WT and H/H pre-cDC2s after 18 hours of culturing in the presence of Flt3L. (c) Frequencies of IRF8hi pre-cDC2s after 18 h of culturing. Data are pooled from three independent experiments (n = 5 for WT and H/H mice). Statistical significance was determined by unpaired, two-tailed Student’s t test. (d) Representative flow cytometry plots showing splenic cDC populations from WT, Irf4−/−, H/H, and H/H Irf4−/− mice (pre-gate: B220− SiglecH− CD11c+ MHCII+ splenocytes). (e) Frequencies of splenic cDC2s and hybrid DCs from the indicated mice. Data are pooled from five independent experiments. Statistical significance was evaluated using one-way ANOVA with Dunnett’s multiple comparisons test. (f) Representative flow cytometry plot depicting cDCs differentiated from the indicated DC progenitors sorted from WT and H/H Irf4−/− BM and cultured in the presence of Flt3L for four days (pre-gate: B220− SiglecH− CD11c+ MHCII+ cells). (g) Frequencies of cDC1s differentiated from WT and H/H Irf4−/− pre-cDC2s. Data are pooled from three independent experiments. Statistical significance was determined by unpaired, two-tailed Student’s t test. Data in c, e, and g are presented as mean values +/− SD. P values are indicated above the graphs.
Extended Data Fig. 5 The Irf8 + 32H enhancer drives hybrid DC development independently of the Irf8 + 41 kb enhancer.
(a) Diagram illustrating the deletion of the Irf8 + 41 kb enhancer on the Irf8 + 32H/H background using CRISPR/Cas9. (b) Sequence of a 658 bp region surrounding the Irf8 + 41 kb enhancer. Nucleotides deleted in both the Irf8 + 41−/− line23 and the Irf8 + 32H/H + 41−/− line are highlighted in red. Additional nucleotides deleted only in the Irf8 + 32H/H + 41−/− line highlighted in blue. sgRNAs target sites are underlined. (c) Representative flow cytometry plots of BM cells stained for pre-cDC1 (upper panel), and splenocytes stained for cDCs (lower panel) from WT, Irf8 + 41−/− (+41−/−), Irf8 + 32H/+ + 41−/− (+32H/+ + 41−/−), and Irf8 + 32H/H + 41−/− (+32H/H + 41−/−) mice. Chromatin phasing diagrams are displayed above the plots for context. Pre-gates: lin− SiglecH− Flt3+ cells for BM, and B220− SiglecH− CD11c+ MHCII+ cells for spleen. (d) Frequencies of pre-cDC1s as a percentage of lin− SiglecH− BM cells across the indicated genotypes. Data are pooled from three independent experiments (n = 4 for WT, 5 for +41−/−, 5 for +32H/+ + 41−/−, and 4 for +32H/H + 41−/− mice). Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. (e) Frequencies of splenic cDC1s, cDC2s and hybrid DCs across the indicated genotypes. Data are pooled from six independent experiments. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. (f) IRF8 expression in cDC2s and hybrid DCs from the indicated genotypes as determined by intracellular staining. Data shown are representative of two similar experiments. Data in d and e are presented as mean values +/− SD. P values are indicated above the graphs.
Extended Data Fig. 6 An optimized Irf8 + 32 kb enhancer drives convergent differentiation of pre-cDC1 and pre-cDC2 toward a hybrid DC phenotype.
(a) Left panel: heatmap visualization of the bulk RNA-seq data as in Fig. 4e. Shown are the top 464 genes differentially expressed between WT cDC1 and cDC2 identified in Fig. 2f. Right panel: zoomed-in views of selected clusters. (b) Schematics illustrating the differentiation of CDP into cDC1, cDC2, and hybrid DC in WT, H/+, and H/H mice. Cell types are color-coded based on IRF8 and BATF3 expression.
Extended Data Fig. 7 Analyses of intermediately optimized Irf8 + 32 kb enhancers.
(a) Diagrams and sequences of the different Irf8 + 32 kb enhancer alleles generated in this study. (b) Retroviral reporter activities of various Irf8 + 32 kb enhancer constructs in cDC1s obtained from Kit/Flt3L-cultured BM. Shown are enhancer-driven GFP expression in transduced cDC1s (pre-gate: B220− CD11c+ MHCII+ CD24+ Sirpa− Thy1.1+ cells). Numbers are GFP geometric MFI. Data shown are pooled from two independent experiments (n = 3 for WT mice). Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison test. (c) Representative flow cytometry plots of splenocytes stained for cDCs (pre-gate: B220− SiglecH− CD11c+ MHCII+ splenocytes). (d) Frequencies of the indicated DC subsets as percentages of B220− SiglecH− CD11c+ MHCII+ cDCs. Data are pooled from six independent experiments. Statistical significance was evaluated using one-way ANOVA with Dunnett’s multiple comparisons test. (e) Representative flow cytometry plots of CD11c-enriched splenocytes stained for cDCs (pre-gate: B220− CD11c+ MHCII+ splenocytes). (f) Frequencies of the indicated DC subsets as percentages of B220− CD11c+ MHCII+ cDCs. Data are pooled from two independent experiments (n = 4 for WT and I/I mice). Statistical significance was determined by unpaired, two-tailed Student’s t test. (g) Representative histograms showing intracellular IRF8 levels in DC subsets gated in (e). Numbers are geometric MFI. (h) IRF8 geometric MFI in cDC1s gated in (e). Statistical significance was determined by unpaired, two-tailed Student’s t test. Data shown are pooled from two independent experiments (n = 4 for WT and I/I mice). Data in b, d, f, and h are presented as mean values +/− SD. P values are indicated above the graphs.
Extended Data Fig. 8 scRNA-seq analysis of WT and Irf8 + 32H/H BM and spleen.
(a) Clustering of BM lin− Flt3+ Kitint-lo cells from WT and H/H mice projected onto a UMAP space (22,839 total cells, with 11,441 from WT and 11,398 from H/H mice). (b) Distributions of WT (blue) and H/H (red) cells across the UMAP in (a). (c) Dot plot of top 10 differentially expressed genes for each cluster in (a). (d) Clustering of BM lin− Flt3+ Kitint-lo cells in (a) split by individual biological replicates. (e) Normalized and scaled Irf8 expression in WT (upper panel) and H/H (lower panel) cells. (f) Violin plot of normalized and scaled Irf8 expression across clusters. WT (blue) and H/H (red) cells are displayed side by side for each cluster. Statistical significance was determined by a two-tailed Wilcoxon rank sum test, applying a log2 fold-change threshold of 0.1. P values are indicated above the graphs for statistically significant comparisons. (g) Clustering of splenic CD11c+ cells from Fig. 5a split by individual biological replicates. (h) Normalized and scaled expressions of selected cluster marker genes in splenic CD11c+ cells.
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Supplementary Fig. 1: Gating strategies for flow cytometry.
Supplementary Tables 1–4
Supplementary Table 1: EMSA probes and competitors. Supplementary Table 2: Enhancer variants utilized in retroviral reporter assays. Supplementary Table 3: Antibodies used for flow cytometry. Supplementary Table 4: gRNAs, ssODN and genotyping primers related to the new mouse strains.
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Ou, F., Liu, TT., Desai, P. et al. Optimization of the Irf8 +32-kb enhancer disrupts dendritic cell lineage segregation. Nat Immunol 25, 2043–2056 (2024). https://doi.org/10.1038/s41590-024-01976-w
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DOI: https://doi.org/10.1038/s41590-024-01976-w
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