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Distinct progenitor lineages contribute to the heterogeneity of plasmacytoid dendritic cells

Abstract

Plasmacytoid dendritic cells (pDCs) are an immune subset devoted to the production of high amounts of type 1 interferons in response to viral infections. Whereas conventional dendritic cells (cDCs) originate mostly from a common dendritic cell progenitor (CDP), pDCs have been shown to develop from both CDPs and common lymphoid progenitors. Here, we found that pDCs developed predominantly from IL-7R+ lymphoid progenitor cells. Expression of SiglecH and Ly6D defined pDC lineage commitment along the lymphoid branch. Transcriptional characterization of SiglecH+Ly6D+ precursors indicated that pDC development requires high expression of the transcription factor IRF8, whereas pDC identity relies on TCF4. RNA sequencing of IL-7R+ lymphoid and CDP-derived pDCs mirrored the heterogeneity of mature pDCs observed in single-cell analysis. Both mature pDC subsets are able to secrete type 1 interferons, but only myeloid-derived pDCs share with cDCs their ability to process and present antigen.

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Fig. 1: pDCs develop primarily from IL-7R+ lymphoid progenitors.
Fig. 2: SiglecH+Ly6D+IL-7R+ LPs have exclusive pDC potential.
Fig. 3: SiglecH+Ly6D+ DP cells are bona fide pDC progenitors.
Fig. 4: Stage-specific transcriptional signatures define pDC commitment.
Fig. 5: Expression of IRF8 marks pDC lineage commitment on SP cells.
Fig. 6: IRF8 and EBF1 define pDC and B cell lineage dichotomy.
Fig. 7: Single-cell analysis elucidates pDC heterogeneity.
Fig. 8: Functional heterogeneity of pDCs is developmentally encoded.

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Acknowledgements

We thank A. G. Rolink (DBM University of Basel), K. M. Murphy (Washington University in St. Louis), and P. Tsapogas (DBM University of Basel) for sharing reagents, expertise and discussions; B. Lambrecht and M. Guilliams (VIB-UGent Center for Inflammation Research) for sharing data and reagents; M. Busslinger (Research Institute of Molecular Pathology IMP, Vienna) for kindly providing us with Ebf1hCD2 reporter mice; D. Schreiner and C. King for constant input and critical reading of the manuscript; D. Labes (DBM University of Basel), K. Eschbach and C. Beisel (Department of Biosystems Science and Engineering (D-BSSE) University of Zurich, Basel) for excellent technical support; the DBM-Microscopy Core Facility; A. Brülhart and all the animal caretakers of WRO1060; and C. Cannavo for IT support. This study was supported by SNF project number PP00P3_150714 and by the Novartis Foundation for medical-biological research n.16A052. This work is dedicated to the memory of Ton Rolink.

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P.R.F. and R.T. designed the project, performed experiments, interpreted the data and wrote the manuscript. L.A.-S. and R.I. analyzed the RNA-sequencing data and contributed to writing the paper. A.E. and G.E.G.-R. performed experiments. All authors agreed to the submission of the manuscript for publication.

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Correspondence to Roxane Tussiwand.

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Integrated supplementary information

Supplementary Figure 1 pDCs develop primarily from IL-7R+ lymphoid progenitors.

(a-c) CDPs, IL-7R+ LP and CSF1RIL-7R NP progenitors as gated in Fig. 1a were cultured over 8 days in the presence of FLT3L (b) or FLT3L and OP9 stromal cells (a,c). Shown are two color histograms for the expression of SiglecH and CD19 at day 4 of culture (a). Relative cell output for pDCs (SiglecH+ CD317+CD19), cDC1 (SiglecHCD317CD19CD11c+MHCII+CD24+CD11b), cDC2 (SiglecHCD317CD19CD11c+MHCII+CD24CD11b+) and B cells (CD19+) was determined at day 4, 6 and 8 as indicated (n=6 independent experiments, thin line represents the mean +/-s.d.). (d-l) Sort purified progenitors as defined in methods were isolated from CD45.1 and CD45.2 mice and used in a 1:1 ratio for in vitro or in vivo experiments as explained in Supplementary Fig. 1i. (d,e) Shown are representative two-color histograms pre-gated on SiglecH+CD11c+ pDCs (d) or pre-gated on SiglecHCD11c+MHCII+ cDCs (e) of IL-7R+ LP (CD45.1) and CDPs (CD45.2) cultured over 8 days in the presence of FLT3L. (n=3 independent experiments). (f) Shown are representative two-color histograms for the expression of CD45.1 and CD45.2 pre-gated on CD19+ B cells of progenitor subsets, as indicated co-cultured for 4 days in the presence of FLT3L and OP9 stromal cells. (g,h) Percent output of CD45RA+ SiglecH+ pDCs (g) and CD19+ B cells (h) of progenitors cultured as in (f) (n=3 independent experiments, each dot represents a mouse, thin line represents the mean +/-s.d.). (j-k) Bone marrow and splenic SiglecH+CD317+pDC output was determined 4 days after in vivo i.v. co-transfer of CD45.1 and CD45.2 LinB220Ly6CCD117hiCD135+ (c-kithi) progenitors. Shown are representative two-color histograms for the expression of CD45.1 and CD45.2 (j) or percent donor derived pDCs in BM and spleen (k) (n=3 independent experiments). (l) CD19+ BM B cells and splenic SiglecHCD317CD19CD11c+MHCII+ cDC output was determined 4 days after in vivo i.v. co-transfer of CD45.1 CDPs and CD45.2 IL-7R+ LP (n=6 independent experiments). Shown are representative two-color histograms for the expression of CD45.1 and CD45.2. Statistical analysis was done using two-way ANOVA with Tukey post-test (g,h,k) (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).

Supplementary Figure 2 SiglecH+Ly6D+ IL-7R+ LPs have exclusive pDC potential.

(a) Shown are box plots (median, range and 25th/75th percentile) for the frequency in the bone marrow of C57BL/6 mice of progenitors and mature CD45RA+CD317+ pDCs as indicated (n=6 independent experiments). (b-j) Sort-purified CD317+B220+CD11c+CCR9 (CCR9), LinB220Ly6CCD117highCD135+ (c-kithi), CDPs, CSF1RIL-7R NP, IL-7R+ LP and DP precursors, as defined in Methods, were cultured for 4 days in the presence of FLT3L (b-e) or in the presence of FLT3L and OP9 stromal cells (f-j). Shown are representative two-color histograms for the indicated markers (b,d,f,g), the total output (c,h,i) and the percent (e,j) mature CD45RA+CD317+ pDCs, SiglecHCD317CD19CD11c+MHCII+CD24+CD11b cDC1, SiglecHCD317CD19CD11c+MHCII+CD24CD11b+ cDC2 and CD19+ B cells derived from the indicated progenitors (n=3 (d,e,g,j) n=5 (b,c,f,h,i) independent experiments, thin line represents the mean +/-s.d.). Statistical analysis was done using one-way ANOVA with Tukey post-test (c,h,i) (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).

Supplementary Figure 3 SiglecH+Ly6D+ DP cells are bona fide pDC progenitors.

(a) Shown are single-color histograms for the indicated markers expressed by DP progenitors (blue), CDPs (green), mature pDCs (gray) (n=3 independent experiments). (b) IFN-α was measured on supernatant collected 16h after stimulation with CpG-A from DN or SP progenitor cultured over-night (day 0) or after 4 days in the presence of FLT3L, as indicated (n=3 independent experiments, shown is a representative experiment. Each dot represents a technical replicate, thin line represents the mean +/-s.d.). (c) May-Gruenwald staining on cytospins of ex-vivo sort-purified DN and SP progenitors as described in Methods (8 representative images taken from 3 independent experiments, scale=10 um). (d,e) DP (d) and SP (e) progenitors were sorted as described in Methods and cultured over 5 days in the presence of FLT3L. Shown are representative two-color histograms for the expression of CD45RA and SiglecH at the indicated time points (n=3 independent experiments).

Supplementary Figure 4 Stage-specific transcriptional signatures define pDC commitment.

(a-f) RNASeq was performed on sort purified DN, SP, DP and mature BM pDCs as described in Methods. (a,b) Shown are developmental heatmaps, as described in Fig 4c, for transcription factors with expression values log2FC >1.5 (a), or cell-surface markers with expression values log2FC > 3 (b). (c) Shown is Gene-Set Enrichment Analysis (GSEA) performed on Hallmark Signatures comparing DP pre-pDC progenitors with mature pDCs. (d-f) Volcano plots showing pair-wise comparisons of DP pre-pDC against DN, SP and mature pDC transcripts as indicated. Depicted in red are up-regulated transcripts (log2FC > 1), in blue down-regulated transcripts (log2FC < -1). (n=4 independent experiments. For each experiment, all progenitors were obtained from one mouse).

Supplementary Figure 5 Expression of IRF8 at the SP stage marks pDC lineage commitment.

(a) Shown are percent mature BM and splenic pDCs, DN, SP and DP progenitors, gated as described in Methods present in C57BL/6, Flt3L−/− and IL-7−/− mice. (b) QRT-PCR of the indicated genes on sort purified DN, SP and DP progenitors, gated as described in Methods, SiglecH+B220+Ly6D+Ly6C+ BM mature and SiglecH+CD317+CD11c+ splenic mature pDCs, splenic CD11c+MHCIIhiCD24+XCR1+ cDC1 and CD11c+MHCIIhi CD11b+Sirp-a+ cDC2. (n=3 independent experiments, shown are mean values of representative results, dots represent technical replicates +/-s.d.). (c-f) C57BL/6, Flt3l−/− and IL-7−/− mice were analyzed. Shown are representative two-color histograms (c-e) for the indicated markers and total splenic CD19+ B cells, CD11c+MHC-IIhiCD24+CD11b cDC1, and CD11c+MHC-IIhiCD24CD11b+ cDC2 (f) of mice of the indicated genotype. (c-d) DN (black), SP (red) and DP (blue) progenitors, pre-gated as described in Methods were analyzed for the expression of IRF8 and EBF1 (right panels) in Flt3l/ (c) and IL-7/ (d). ((a,c-f) n=6 independent experiments. (f) Individual mice are plotted, mean +/- s.d). Statistical analysis was done using two-tailed Student’s t test (f) (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).

Supplementary Figure 6 IRF8 and EBF1 define pDC and B cell lineage dichotomy.

(a,c) Shown are gating strategies for DN, SP and DP progenitors in IRF8-eGFP (a) and Ebf1-hCD2 (c) reporter mice (n=3 independent experiments, shown is a representative experiment). (b,d) Shown is the total output of CD45RA+CD317+ pDCs (b) or CD19+ B cells (d) per plated progenitor cell cultured in the presence of FLT3L (b) or FLT3L and OP9 stromal cells (d) (n=3 independent experiments, shown is mean +/-s.d.). (e-h) Shown are two color histograms (e,g) for the expression of CD45RA/CD317 (e) and SiglecH/CD19 (g) of sort-purified SP progenitors from C57BL/6, Ebf1 SP and Ebf1+ SP progenitors, from Ebf1-hCD2 reporter mice as indicated culture for 4 days in the presence of FLT3L (e,f) or FLT3L with OP9 stromal cells. (g,h) Shown are mean +/-s.d. for percent CD45RA+CD317+ pDCs (f) and CD19+ B cells (h) obtained as described in e and g (n=3 independent experiments). (i-l) Shown are two color histograms for the expression of CD317/CD19 (i, k), and percent CD317+CD45RA+ pDCs and CD19+ B cells (j, l) of DP (i,j) and SP (k,l) progenitors isolated as described in Methods from C57BL/6 mice and cultured for 4 days in regular (left panels) or transwell® culture plates (middle and right panels). Progenitors cultured in transwell® were plated either in direct contact with OP9 stromal cells (lower chamber) or on the upper chamber, as indicated. (n=5 independent experiments. Shown are mean +/-s.d.). Statistical analysis was done using one-way ANOVA with Tukey post-test (b,d,h) or two-tailed Student’s t test (j,l) (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001).

Supplementary Figure 7 Single-cell analysis unravels pDC heterogeneity.

Single cell RNA sequencing delineates heterogeneous clusters within mature pDCs. (a-d) RNA Sequencing was performed as described in Methods on mature BM and splenic pDCs, and on in vitro generated pDCs from IL-7R+ LPs and CDPs. (a) Hierarchical clustering of all samples based on Pearson's correlation coefficient calculated on 25% of genes with highest variance (calculated as inter-quartile range). (b) Shown is the average expression level across all samples for the indicated genes. (c-d) Shown are relative expression levels for differentially expressed transcription factors (c) or cell-surface genes (d) (log2FC >1.5). (e) The 8 clusters, identified from the single cell RNA sequencing analysis (Fig. 7d and 7e) are plotted as percentage. (f) Shown is the number differentially expressed genes (DEG) (Log2FC>abs(1.5)) of the identified clusters as a pairwise comparison. (g) The total number of detected genes is plotted for each cluster as a violin plot (median, range and 25th/75th percentile). (h) PCA analysis for the identified clusters for genes related to G1/G0, G2 and S phase. Cells for bulk and single cell RNASeq were harvested from 3 mice on three independent experiments.

Supplementary Figure 8 Phenotypic characterization of pDC-like cells.

(a-f) (a) Shown are the gating strategies for CD317+SiglecH+Zbtb46-GFP pDCs (red) and CD317+SiglecH+Zbtb46-GFP+ pDC like cells (blue) pre-gated on CD3CD19 cells (n=5). (b,d,f) Single cell analysis of BM and splenic pDCs as described in Fig. 7. Shown is the relative expression level for the indicated genes. The size of each dot corresponds to the relative expression of a given gene for each cell. Contour lines indicate density of the BM (blue) and splenic (red) cells in the PCA space. (c,e) Single-color histograms for the indicated surface markers expressed by BM (empty) or splenic (full) pDCs (red) and pDC like cells (blue) gated as in a. (n=3).

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Supplementary Figures 1–8 and Supplementary Information Note

Reporting Summary

Supplementary Table 1

Stage-specific transcriptional signatures

Supplementary Table 2

Stage-specific Gene Ontology (GO) enrichment analysis

Supplementary Table 3

Top hit genes for each cluster

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Rodrigues, P.F., Alberti-Servera, L., Eremin, A. et al. Distinct progenitor lineages contribute to the heterogeneity of plasmacytoid dendritic cells. Nat Immunol 19, 711–722 (2018). https://doi.org/10.1038/s41590-018-0136-9

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