Abstract
Mouse conventional dendritic cells (cDCs) can be classified into two functionally distinct lineages: the CD8α+ (CD103+) cDC1 lineage, and the CD11b+ cDC2 lineage. cDCs arise from a cascade of bone marrow (BM) DC-committed progenitor cells that include the common DC progenitors (CDPs) and pre-DCs, which exit the BM and seed peripheral tissues before differentiating locally into mature cDCs. Where and when commitment to the cDC1 or cDC2 lineage occurs remains poorly understood. Here we found that transcriptional signatures of the cDC1 and cDC2 lineages became evident at the single-cell level from the CDP stage. We also identified Siglec-H and Ly6C as lineage markers that distinguished pre-DC subpopulations committed to the cDC1 lineage (Siglec-H−Ly6C− pre-DCs) or cDC2 lineage (Siglec-H−Ly6C+ pre-DCs). Our results indicate that commitment to the cDC1 or cDC2 lineage occurs in the BM and not in the periphery.
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Acknowledgements
We thank L. Robinson for critical review and editing of the manuscript; M.L. Ng, S.H. Tan, T.B. Lu, I. Low and N.B. Shadan for technical assistance; K. Murphy (University of Washington) for ZBTB46-GFP BM; and G. Belz (Walter & Eliza Hall Institute) for Id2-GFP BM. Supported by Singapore Immunology Network (F.G.), Agency for Science, Technology and Research (BMRC Young Investigator grant to A.S.), the Genomics Institute of Singapore (P.R.) and the Singapore Medical Research Council (NMRC/CBRG/0047/2013 to B.M.).
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A.S., P.R. and F.G. conceived of the study; A.S., S.V., H.R.B.S., J.S., J.L. and B.M. performed experiments; A.S., J.C., S.Z., M.P. and F.G. analyzed data; A.L., F.Z., L.R., S.N. and E.W.N. provided reagents and intellectual guidance; and A.S., S.V., J.C. and F.G. wrote the paper.
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Integrated supplementary information
Supplementary Figure 1 Single-cell transcriptomic workflow and quality control.
Experimental approach for analysis of gene expression in single MDPs, CDPs and pre-DCs using the Fluidgim C1 autoprep system (a). Box plot of log2 r.p.k.m. values of the 264 most commonly expressed genes in single MDPs, CDPs and pre-DCs. Single MDPs, CDPs and pre-DCs were ranked according to their median values of the gene expression of these 264 genes from high to low. The dashed horizontal line in red indicates the threshold used to predict the outlier cells. Red boxes indicate outlier cells that had median log2 r.p.k.m. values below the threshold due to poor sequencing efficiency (b). Principal component Analysis (PCA) plot of cells using the 264 most commonly expressed genes. Red boxes indicate outlier cells that were identified in Supplementary Fig. 1b (c). Bar plot of total number of reads, number of mapped reads and mapping rate in each individual MDP, CDP and pre-DC (d). Correlation of ERCC spike-in RNA and detected r.p.k.m values detected across all mRNA sequenced cells (R2: Pearson’s correlation coefficient, (e)). Assessment of capture batch effect on CDP single cell mRNA sequencing data. CDP color coded for different capture batches analyzed using hierarchical clustering (f). Assessment of capture batch effect on pre-DC single cell mRNA sequencing data. pre-DC color coded for different capture batches analyzed using hierarchical clustering (g). MDP was captured using 1 Fluidigm C1 IFC, CDP was captured using 5 Fluidigm C1 IFCs, pre-DC was captured using 3 Fluidigm C1 IFCs.
Supplementary Figure 2 Comparison of different sequencing depths in single CDP transcriptomes.
47 CDP libraries were sequenced to a read depth between 10 – 15 million reads per cell and compared to their counterparts already sequenced to 1 – 2 million reads per cell (a). Additionally, the number of expressed genes detected per single CDP was compared between the deeper sequencing depth and the depth used throughout our analysis (b). Hierarchical clustering of all MDPs, CDPs and pre-DCs based on all expressed genes (r.p.k.m > 1) before selection of the Top 87 PCA-loaded genes determined by ANOVA (c).
Supplementary Figure 3 Unsupervised clustering of the DC development continuum.
Heatmap depicts unsupervised clustering of Top 87 PCA-loaded genes, determined by ANOVA across all transcriptomes of single MDP, CDPs and pre-DCs (ANOVA followed by multiple test correction using the Benjamini & Hochberg method). This heatmap was then reprocessed with the NBOR algorithm (see methods) to generate the heatmap in Fig. 1a. Assigned landmarks depicted under the heatmap, genes can be found in Supplementary Table 2 (z-score normalized, (a)). Bioinformatics workflow for ordering single cells along a linear development continuum using the neighborhood-based ordering strategy (b).
Supplementary Figure 4 Workflow for identification of cDC1 lineage– or cDC2 lineage–primed progenitors using CMap.
Workflow depicts strategy to identify total DC, cDC1 or cDC2 lineage-primed progenitors using transcriptomic signatures derived from conventional transcriptome analysis combined with CMap analysis.
Supplementary Figure 5 Characterization of novel pre-DC subsets defined by expression of Siglec-H and Ly6C in mouse BM.
Flow cytometric analysis of CD24 expression on Siglec-H+Ly6C−, Siglec-H+Ly6C+, Siglec-H−Ly6C− or Siglec-H−Ly6C+ pre-DC subsets in BM, blood and spleen (representative data shown of 3 independent experiments with a total of 7 individual mice, (a)) (a). Representative scanning electron microscopy images of individual cells from each pre-DC subset following purification from the bone marrow by FACS (b). Flow cytometric analysis of intensity of GFP expression in ZBTB46, ID2 and CX3CR1 reporter mice in Siglec-H+Ly6C−, Siglec-H+Ly6C+, Siglec-H−Ly6C− or Siglec-H−Ly6C+ pre-DC subsets from mouse BM, number depicts mean fluorescent intensity (MFI) (representative data shown of 3 independent experiments with a total of 2 mice per strain per experiment, (c)). Frequency of pre-DC subsets among total pre-DCs in BM, blood and spleen (data representative of 3 independent experiments with 3 mice per experiment, mean ± SEM, (d)). Flow cytometric analysis of Ly6C and Siglec-H expression on the progeny of CD45.2+ pre-DC subsets (CD45.2+Siglec-H+Ly6C−, CD45.2+Siglec-H+Ly6C+, CD45.2+Siglec-H−Ly6C+ or CD45.2+Siglech-H−Ly6C− pre-DCs) added individually into day 2 in vitro CD45.1+ FLT3L-stimulated BM cultures. Cells depicted were harvested on day 3 and gated for Lin−B220−MHCII−CD45.2+CD11c+ expression (data representative of 2 independent experiments with 1 analyte per population, mean ± SEM, (e)).
Supplementary Figure 6 Identification of subset-specific DC progenitors in vitro and in vivo.
Percentage of proliferating cells among total Siglec-H+ BM pre-DC subsets and total Siglec-H− BM pre-DCs as determined by expression of Azami green expression in Fucci mice measured in vivo (data representative of 3 independent experiments with 7 mice in total, mean ± SEM, p: 0.0003, unpaired, two-tailed t-test, *p < 0.001, (a)). Flow cytometric analysis of B220 and MHC II expression on the progeny of CD45.2+ pre-DC subsets (CD45.2+Siglec-H+Ly6C−, CD45.2+Siglec-H+Ly6C+, CD45.2+Siglec-H−Ly6C+ or CD45.2+Siglech-H−Ly6C− pre-DCs) added individually into day 2 in vitro CD45.1+ FLT3L-stimulated BM cultures. Cells depicted were harvested on day 3 or 6 and are gated CD45.2+CD11c+ expression (data representative of 3 independent experiments with 1 analyte per population (b)). Pie charts depict flow cytometric analysis of single Siglec-H+Ly6C− pre-DC or Siglec-H+Ly6C+ pre-DC progeny cultured for 6 days on OP9 feeder cells in day 9 FLT3L-stimulated BM culture conditioned media. Progeny of single Siglec-H+Ly6C− pre-DC or Siglec-H+Ly6C+ pre-DC was analyzed for the presence of pDC, cDC1 and cDC2 (n depicts number of clones analyzed, Siglec-H+Ly6C− pre-DC n=24, Siglec-H+Ly6C+ pre-DC n=35 (c)). Flow cytometric analysis of phenotypes of splenic progeny from CD45.2+Siglec-H+Ly6C−, CD45.2+Siglec-H+Ly6C+, CD45.2+Siglec-H−Ly6C+ or CD45.2+Siglech-H−Ly6C− pre-DCs injected into femurs of recipient mice 5 days earlier (data representative of 3 independent experiments with 1 analyte per pre-DC population, (d)). Schematic depiction of the DC development cascade in the bone marrow; CDPs as well as Siglec-H+Ly6C− pre-DCs show potential to give rise to cDCs as well as pDCs, while acquisition of Ly6C on Siglec-H+Ly6C− pre-DCs leads to the loss of pDC potential. Siglec-H+Ly6C+ pre-DCs are further differentiating in Siglec-H−Ly6C+ as well as Siglec-H−Ly6C− pre-DC with selective potential for cDC2 or cDC1 respectively (e).
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Schlitzer, A., Sivakamasundari, V., Chen, J. et al. Identification of cDC1- and cDC2-committed DC progenitors reveals early lineage priming at the common DC progenitor stage in the bone marrow. Nat Immunol 16, 718–728 (2015). https://doi.org/10.1038/ni.3200
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DOI: https://doi.org/10.1038/ni.3200
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