Plasmacytoid dendritic cells develop from Ly6D+ lymphoid progenitors distinct from the myeloid lineage

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Abstract

Dendritic cells (DC) are currently classified as conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Through a combination of single-cell transcriptomic analysis, mass cytometry, in vivo fate mapping and in vitro clonal assays, here we show that, at the single-cell level, the priming of mouse hematopoietic progenitor cells toward the pDC lineage occurs at the common lymphoid progenitor stage, indicative of early divergence of the pDC and cDC lineages. We found the transcriptional signature of a pDC precursor stage, defined here, in the IL-7Rα+ common lymphoid progenitor population and identified Ly6D, IL-7Rα, CD81 and CD2 as key markers of pDC differentiation, which distinguish pDC precursors from cDC precursors. In conclusion, pDCs developed in the bone marrow from a Ly6DhiCD2hi lymphoid progenitor cell and differentiated independently of the myeloid cDC lineage.

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Fig. 1: Pre-DC heterogeneity reveals distinct pDC and cDC progenitors.
Fig. 2: CD115 CDPs are highly primed for pre-pDCs.
Fig. 3: Heterogeneity among early progenitors reveals early priming for pre-cDC versus pre-pDC differentiation.
Fig. 4: Ly6D expression defines early lymphoid pDC progenitors.
Fig. 5: Ly6D+CD115 CDPs and Ly6D+ CLPs are early pDC-committed progenitors.
Fig. 6: CD2 expression identifies pDC-committed progenitors.
Fig. 7: Unbiased reconstruction of BM progenitors supports pDC differentiation.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. The single-cell RNA-seq data generated in the current study are available in the NCBI Gene Expression Omnibus database under accession code GSE130966.

Change history

  • 04 July 2019

    The Supplementary Information file initially published online was corrupted and was replaced on 4-Jul-2019.

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Acknowledgements

We thank L. Robinson of Insight Editing London for critical review and editing of the manuscript. F.G. is an EMBO YIP awardee and is supported by Singapore Immunology Network (SIgN) core funding, as well as Singapore National Research Foundation Senior Investigatorship (NRFI) NRF2016NRF-NRFI001-02. The CyTOF, bioinformatics and immunogenomics platforms are part of the SIgN Immunomonitoring platform (supported by a BMRC IAF 311006 grant and BMRC transition funds H16/99/b0/011).

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R.J.D., A.S., I.L., N.B.S., A.T., A.G., Y.Y.H., J.L. and M.F.B.M.K. performed experiments. R.J.D., C.-A.D., A.G., E.B., Y.C., J.C., E.W.N., M.C. and F.G. analyzed data. J.C., A.L., E.W.N. and I.A. provided intellectual guidance. R.J.D. and F.G. wrote the paper. F.G. conceptualized the study.

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Correspondence to Florent Ginhoux.

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Integrated supplementary information

Supplementary Figure 1 Workflow and Quality Control of sorted single pre-DCs and pre-DC subsets.

a Experimental approach for analysis of gene expression in single pre-DCs and pre-DC subsets (as identified by expression of SiglecH and Ly6C) using the Fluidigm C1 autoprep system. b Violin plot and c bar plot for number of genes detected in each single cell. Violin plots for nUMI d and percentage of mitochondrial genes e in the sorted single cell populations. f Workflow for CMap analysis of pDC- and cDC-primed single cells using transcriptomic signatures derived from conventional transcriptome analysis combined with CMap analysis. g CMap analysis of BM pDCs, BM pre-DCs, and BM pre-DC subsets before trimming. a–g Data are representative for one experiment with 96 single BM pre-DCs, 16 single blood pre-DCs 70 single SiglecH-Ly6C-, 68 single SiglecH-Ly6C+, 70 single SiglecH+Ly6C-, and 45 single SiglecH+Ly6C+ pre-DCs and 26 single pDCs.

Supplementary Figure 2 Workflow and Quality Control for sorted single CDP populations.

a Experimental approach for analysis of gene expression in single CD115- CDPs and single CD115+ CDPs using the Fluidigm C1 autoprep system. Violin plots for b number of genes detected in each single cell, c nUMI and d and percentage of mitochondrial genes in the sorted single cell populations. e Bar plot of number of expressed genes in individual sorted single cells. f CMap analysis of enrichment of pDC-signature genes versus cDC-signature genes in CD115- CDPs and CD115+ CDPs. a–f Data are representative for one experiment with 96 single BM CD115+ CDPs and 58 single CD115- CDPs. g Quantification of the cDC and pDC progeny of cultured CD115+ CDPs on day 7 of culture with rh-Flt3L. h Flow cytometry analysis of the progeny of sorted, cultured Ly6D+CD81-, Ly6D+CD81+, Ly6D-CD81+, and Ly6D-CD81- CD115- CDPs on day 7 of in vitro stimulation with rh-Flt3L. g,h Data are representative for three experiments with one replicate per culture condition. i Percentage of Ly6D+CD81-, Ly6D+CD81+, Ly6D-CD81+, and Ly6D-CD81- CD115- CDPs and SiglecH-/+ pre-pDCs in the BM. j Percentage, absolute numbers and percentage of apoptotic BM LMPP, CMP, GMP, Ly6D- CLP, Ly6D-CD115- CLP, Ly6D- pre-pDC, CD115+ CDP, Ly6D+CD115- CDP, Ly6D+ pre-pDC and Ly6D+ pre-cDC. k Percentage, absolute numbers and percentage of apoptotic BM pDCs. l Percentage of proliferating cells among analyzed cell populations. m Flow cytometry analysis of percentage of Annexin V vs. DAPI stained cells among the indicated cell populations. n Flow cytometry analysis of percentage of Annexin V vs. DAPI stained BM pDCs. h–n Mean ± s.e.m. Data are representative for three independent experiments with three mice each.

Supplementary Figure 3 Workflow and Quality Control for sorted single BM progenitors.

a Experimental approach for analysis of gene expression in single CLPs, GMPs, LMPPs, and MDPs using the Fluidigm C1 autoprep system. Violin plots for b number of genes detected in each single cell, c nUMI and d and percentage of mitochondrial genes in the sorted single cell populations. e Bar plot of number of expressed genes in individual sorted single cells. Data are representative for one experiment with 63 single LMPPs, 51 single GMPs, 48 single CLPs, 59 single MDPs. f Flow cytometry analysis of surface molecule expression of CD81 and Ly6D on Ly6C-SiglecH- pre-DCs, Ly6C+SiglecH- pre-DCs, SiglecH+Ly6C+ pre-DCs, and SiglecH+Ly6C- pre-DCs. Data are representative of three independent experiments with three mice. g Venn diagram comparison of pre-pDC primed DEGs shared between CLPs and SiglecH+Ly6C- pre-DCs for g least 50 % or h 10% of cells per population. g,h Data are representative for one experiment with 48 single CLPs, and 70 single SiglecH+Ly6C- pre-DCs.

Supplementary Figure 4 UMAP analysis of the murine BM compartment.

a UMAP analysis of CyTOF data of murine BM cells. Mayor cell populations as indicated in the UMAP plot (and by the key on the right margin) and relative expression of selected markers among the total BM cell compartment. Data are representative for one experiment with 5 mice.

Supplementary Figure 5 Progeny of CLPs on day 5.

a tSNE analyses (as in Fig. 5) for progeny of index-sorted single CD115 CDPs showing their expression levels of CD81 and Ly6D and analyses of pDC vs. cDC offspring for index-sorted single Ly6D+CD81 (identified with “1”) or Ly6D+CD81+ (identified with “2”) or Ly6DCD81+ (identified with “3”) or Ly6DCD81 (identified with “4”) CD115 CDPs. Data are representative for one experiment with 48 single cells per sorted CDP population. b Progeny of index-sorted single CLPs on day 5 of co-culture with OP9 stromal feeder cells in complete medium supplemented with rh-Flt3L (pDC progeny = red; cDC progeny = blue; bipotent progeny = yellow; no progeny = grey). Data are representative for one experiment with 96 single CLPs. c Percentage of pDC or cDC progeny from transferred sorted CD45.2+ BM progenitors in BM and spleen on day 14 post transfer, d percentage of T cell- NK cell- and ILC progeny from transferred sorted CD45.2+ BM progenitors in spleen and thymus on day 7 post transfer, e percentage of neutrophil progeny from transferred sorted CD45.2+ BM progenitors in blood, BM and spleen on day 7 post transfer and f percentage of pDC and B cell progeny from transferred sorted CD45.2+ BM progenitors in BM and spleen on day 7 post transfer. c–f Mean ± s.e.m. Data are representative of six experiments with one mouse per group for day 7, for three experiments with one mouse per group for day 3, and for three experiments with one mouse per group for day 14.

Supplementary Figure 6 Workflow and Quality Control of sorted single pre-pro B cells.

a Experimental approach for analysis of gene expression in single pre-pro B cells using the Fluidigm C1 autoprep system. Violin plots for b number of genes detected in each single cell, c nUMI and d and percentage of mitochondrial genes in the sorted single cell populations. e Bar plot of number of expressed genes in individual sorted single cells. f Expression of Rag1 in single BM progenitor cells. Data are representative for one experiment with 26 single pre-pro B cells.

Supplementary Figure 7 pDC ontogeny revised.

a A heatmap of the top differentially expressed genes from the reference map of 8,395 bone marrow myeloid progenitor cells. b Expression of important lineage markers along the differentiation axis from LMPPs to pDCs. Color-bar indicate cluster association as in Fig. 7e. Data are representative of one experiment with 8,395 bone marrow myeloid progenitors and 63 single LMPPs, 51 single GMPs, 48 single CLPs, 59 single MDPs, 96 single CD115+ CDPs, 58 single CD115 CDPs, 96 single BM pre-DCs, 16 single blood pre-DCs 70 single SiglecHLy6C, 68 single SiglecHLy6C+, 70 single SiglecH+Ly6C, and 45 single SiglecH+Ly6C+ pre-DCs and 26 single pDCs. c,d Schematic trees of pDC (pILC) differentiation depicting the c current model and d a model highlighting the findings of our study.

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Dress, R.J., Dutertre, C., Giladi, A. et al. Plasmacytoid dendritic cells develop from Ly6D+ lymphoid progenitors distinct from the myeloid lineage. Nat Immunol 20, 852–864 (2019). https://doi.org/10.1038/s41590-019-0420-3

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