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

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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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

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.

Corresponding author

Correspondence to Kenneth M Murphy.

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The authors declare no competing financial interests.

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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