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The activation trajectory of plasmacytoid dendritic cells in vivo during a viral infection


Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN-I). What other functions pDCs exert in vivo during viral infections is controversial, and more studies are needed to understand their orchestration. In the present study, we characterize in depth and link pDC activation states in animals infected by mouse cytomegalovirus by combining Ifnb1 reporter mice with flow cytometry, single-cell RNA sequencing, confocal microscopy and a cognate CD4 T cell activation assay. We show that IFN-I production and T cell activation were performed by the same pDC, but these occurred sequentially in time and in different micro-anatomical locations. In addition, we show that pDC commitment to IFN-I production was marked early on by their downregulation of leukemia inhibitory factor receptor and was promoted by cell-intrinsic tumor necrosis factor signaling. We propose a new model for how individual pDCs are endowed to exert different functions in vivo during a viral infection, in a manner tightly orchestrated in time and space.

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Fig. 1: Bulk transcriptional profiling suggests the induction of distinct pDC activation states in vivo during MCMV infection.
Fig. 2: ScRNA-seq analysis of pDCs from 36-h MCMV-infected mice confirms their heterogeneity and pinpoints LIFR downregulation as a selective marker of IFN-I-producing pDCs.
Fig. 3: ScRNA-seq analysis identifies seven different pDC activation states in vivo during MCMV infection.
Fig. 4: Inference of the pDC activation trajectory during MCMV infection.
Fig. 5: Transcriptional convergence of pDCs toward tDCs over pseudo-time.
Fig. 6: Identification of annotated gene modules regulated along pseudo-time during pDC activation.
Fig. 7: Cell-intrinsic TNF signaling promotes pDC IFN-I production.
Fig. 8: Characterization of the phenotype, function and micro-anatomical location of pDC activation states.

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Data availability

Microarray and scRNA-seq data have been deposited in the Gene Expression Omnibus (GEO) repository under accession codes GSE150664 and GSE151248, respectively. All the other data that support the findings of the present study are available from the corresponding authors upon reasonable request.


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We thank all the staff of the CIML and CIPHE mouse houses for their assistance, as well as the staff of the CIML flow cytometry and imaging (ImagImm) core facilities. We thank L. Spinelli for fruitful discussions on single-cell data analysis and the CIML genomics and bioinformatics platform for their technical and methodological help. Microarray experiments and sequencing for the SS2 datasets were performed by V. Alunni or B. Jost in the GenomEast platform (Strasbourg, France), a member of the France Génomique consortium (ANR-10-INBS-0009), managed by C. Thibault-Carpentier. FB5P-seq library sequencing was performed by HalioDx testing laboratory, Marseille, France. We thank S. Henri (CIML) for the generous gift of OT-II mice and H. Luche for advice on cell and tissue fixation to preserve the YFP signal. This research was funded by grants from the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007-2013, grant no. 281225, SystemsDendritic, to M.D.), the Agence Nationale de la Recherche (ANR) (SCAPIN, grant no. ANR-15-CE15-0006-01, to M.D.), and the Fondation pour la Recherche Médicale (grant no. DEQ20180339172, Equipe Labellisée, to M.D.). We also thank the DCBIOL Labex (ANR-11-LABEX-0043, grant no. ANR-10-IDEX-0001-02 PSL*), the A*MIDEX project (grant no. ANR-11-IDEX-0001-02) funded by the French government’s Investissements d’Avenir program managed by the ANR, and institutional support from CNRS, INSERM, Aix-Marseille Université and Marseille Immunopole. This work was supported by the French National Research Agency through the Investments for the Future program (France-BioImaging, ANR-10-INBS-04). A.A. and R.C. were supported by the DCBIOL Labex. G. Brelurut’s apprenticeship was supported by INSERM.

Author information

Authors and Affiliations



Studies were designed by E.T., M.D., A.A. and T.-P.V.M., with help from A.-C.V and P.M. Experiments were performed by A.A., E.T., M.V., N.C. and N.A., with help from K.N. and G. Bessou. Data were analyzed by A.A., T.-P.V.M., M.V., N.C., M.D. and E.T., with help from C.D., R.C., G. Brelurut, I.C.-M., M.T.-C. and D.T. Critical reagents and advice were provided by B.R. and J.-L.D. The manuscript was written by A.A., T.-P.V.-M., M.D. and E.T. All authors contributed to discussions and editing of the manuscript.

Corresponding authors

Correspondence to Marc Dalod or Elena Tomasello.

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The authors declare no competing interests.

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Peer review information Jamie D. K. Wilson and Ioana Visan were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Validation of the Ifnb1Eyfp reporter mice to track IFN-I-producing pDCs during MCMV infection.

a, IFN-α/β production by pDCs (blue), pDC-like cells (red) and cDCs (black) at 36 h after MCMV infection of Zbtb46GFP reporter mice. Overlaid histograms (bottom right) show CD11c and CD11b expression for the three cell types. The data shown are from one mouse representative of 3 animals from 2 independent experiments. b, YFP expression in pDCs is stable during the biological process examined. Ifnb1EYFP CD45.2+ mice were infected by MCMV. 36 h later, LIFRlo EYFP- or LIFRlo EYFP+ pDCs were sorted by flow cytometry and cultured in vitro for 8 h in CD45.1+ feeder FLT3-L bone marrow cultures. YFP expression was monitored by flow cytometry before and after the culture as indicated. The data shown are from one experiment representative of two independent ones. c–g Around 80% of splenic YFP+ cells are bona fide pDCs at all time points examined during MCMV infection. c, cDCs and pDCs were identified following the gating strategy shown. The analysis was performed after selection of live cells and exclusion of Lin+ cells. d, YFP expression was analyzed in indicated splenic DC populations, isolated from 44 h MCMV-infected Ifnb1Eyfp mice. e, Ccr9 expression was analyzed in splenic cDC1s (red), cDC2s (green) and pDCs (blue) isolated from 36 h (left), 44 h (middle) or 48 h (right) MCMV-infected Ifnb1Eyfp mice. f, Autofluorescence-YFP+ cells were gated in live splenocytes isolated from 44 h MCMV-infected Ifnb1Eyfp mice. The proportions of DCs vs non-DCs (others, grey) in YFP+ cells were analyzed according to the gating strategy shown in (c). g, Summary of the results obtained following the strategy shown in (e) at 36 h (left), 44 h (middle) and 48 h (right) post-infection. For each time point, data (mean ± s.e.m.) are shown for 5 mice from one experiment.

Extended Data Fig. 2 Design and quality control of the SS2 dataset#2.

a, Flow cytometry gating and overall strategy for index sorting of pDCs from one uninfected (UN, top panel) and one 36 h MCMV-infected (bottom panel) Ifnb1Eyfp mice, using LIFR and BST2 expression levels to enrich IFN-IEyfp+ pDCs, for scRNAseq. Numbers in parentheses indicate the total number of cells sorted in each gate. b, t-SNE and cell clustering analysis for the 323 cells that passed quality controls. c, Sorting phenotype projection on the t-SNE space. d, LIFR expression projection on the t-SNE space. Data are expressed as inverse hyperbolic arcsine (asinh) of fluorescence intensity. e, BubbleMap illustrating GSEA results for 8 selected gene sets (columns) in pairwise comparisons between the cell clusters (rows) identified in (b). ND, not determined. f, Heatmap (left) showing mRNA expression levels of representative genes (rows) across the cell clusters (columns) identified in (b). Most of the genes shown were selected due to their contribution to the GSEA results from (e), as informed by the grid on the right of the heatmap where filled cells mean belonging of the gene (row) to a gene set (column). The gene set order and color code on the top of the grid is the same as in panel e. The far right column of the grid (*) corresponds to genes selectively expressed to high levels in plasmocytes. g, Normalized expression of Ccl3 vs the IFN-I meta-gene along pseudo-time. h, Projection on the UMAP space of the predicted induction (red) vs termination (blue) of Ccl3 transcription.

Extended Data Fig. 3 Kinetics of IL-12 production by pDCs during MCMV infection.

a, Frequency (mean ± s.e.m.) of YFP+, IFN+ and IL-12+ pDCs isolated from Ifnb1Eyfp mice at indicated time points after MCMV infection. b, Data from individual animals for the frequencies of IL-12+ and YFP+ cells in pDCs isolated from Ifnb1Eyfp mice at indicated time points, with overlay of mean ± s.e.m. c, Flow cytometry dot plots showing IL-12 vs YFP expression in pDCs isolated from one representative Ifnb1Eyfp mouse for each time point. The data from all panels were analyzed from the same experiments, with 5 mice at 0 h, 7 at 33 h, 10 at 36 h, 5 at 40 h, 3 at 44 h and 3 at 48 h, from one experiment for 44 h and 48 h, or pooled from 2 (resp. 3) independent experiments for 33 h and 44 h (resp. 36 h).

Extended Data Fig. 4 LIFR downregulation enables enrichment from WT C57BL/6 mice of the pDCs engaged in IFN-I.

pDCs were sorted from 36 h MCMV-infected WT C57BL/6 mice, with a protocol including an enrichment of LIFRlo cells to increase the capture efficiency for pDCs engaged in IFN-I production. a, Monocle pseudo-temporal analysis showing bifurcation of the inferred pDC activation trajectory in two major branches, Y53 and Y50. b, Expression of the IFN-I meta-gene along pseudo-time for the Y53 (top) and Y50 (bottom) branches of the pDC activation trajectory. c, LIFR expression along pseudo-time on the pDCs from the common root (empty thin orange circles), Y53 branch (filled orange triangles) and Y50 branch (empty thick dark red circles) of the pDC activation trajectory. d, Expression of individual genes along pseudo-time for the cells from the common root (empty thin orange circles), Y53 branch (filled orange triangles) and Y50 branch (empty thick dark red circles) of the pDC activation trajectory. A polynomial curve was fit to the data for each of the three segments of the trajectory.

Extended Data Fig. 5 Design, quality controls and RNA velocity analysis of the FB5P kinetics dataset.

a, Experimental design. For each time point, splenocytes were isolated from one Ifnb1Eyfp mouse, depleted of Lin+ cells by magnetic sorting and used for index sorting of pDCs using three sorting gates: i) total (bulk) pDCs, ii) LIFRlo pDCs irrespective of their YFP expression, and iii) YFP+ pDCs. FB5P-seq scRNAseq libraries were then prepared. b, Table indicating the total numbers of cells sorted for each time point and sorting gate, the numbers of cells that passed quality control upon data analysis, and the number of bona fide pDC ultimately kept after identification and removal of contaminating cell types. c,d, Identification and removal of contaminating B and pDC-like cells. c, UMAP and clustering analysis. The analysis of the genes differentially expressed across clusters combined with their mining for expression across immune cell types by using the MyGeneSet tool of Immgen enabled identification of contaminating B cells (cluster 7, highlighted in orange). A GSEA analysis performed by using BubbleGUM (not shown) enabled identification of contaminating pDC-like cells (cluster 11, highlighted in red). d, Projection on the UMAP space of the expression of two B cell-specific genes, Jchain and Iglv1, two pDC-like cell-specific genes, Ms4a6b and Vim, and 2 genes selectively expressed at high levels in pDCs, Klk1 and Ly6d. e, Projections of the velocity vector of each pDC in the UMAP space obtained after contaminant removal.

Extended Data Fig. 6 Ex vivo unidirectional transition of pDCs isolated from MCMV-infected mice from a YFP+CCR7- to a YFP+CCR7+ activation state.

Ifnb1Eyfp CD45.2+ mice were infected by MCMV. 36 h later, LIFRlo YFP+ CCR7- pDCs and LIFRlo YFP+ CCR7+ pDCs were sorted and cultured in vitro for 8 h in CD45.1+ feeder FLT3-L bone marrow cultures. CCR7 expression was monitored by flow cytometry before and after the culture, as depicted in the right panel. The data shown are from one experiment representative of two independent ones.

Extended Data Fig. 7 Micro-anatomical locations of splenic YFP+ cells during MCMV infection of Ifnb1Eyfp mice.

a, b, c, Number of IFN-α/β spots per mm2 (a), of YFP+IFN-α/β+ cells/mm2 (b), and of YFP+ cells per mm2 (c), in whole spleen sections from Ifnb1Eyfp mice at indicated time points after MCMV infection. d, Number of YFP+ cells per mm2 residing in the different spleen zones. MZ, marginal zone; RP, red pulp; WP, white pulp. Fifteen individual data points are shown on each graph for each time point, corresponding to quantitation of 5 different whole spleen sections per mouse, from 3 different mice, with overlay of mean±s.e.m.

Extended Data Fig. 8 Proposed model of the spatiotemporal dynamics of splenic pDC activation and functions during MCMV infection.

MCMV initially targets and replicates in stromal cells and/or metallophilic macrophages in the marginal zone (❶). These infected cells may then upregulate their expression of ICAM-1 and express mTNF, leading to their specific recognition by, and interactions with, quiescent pDCs. This interaction is proposed to downregulate LIFR expression on pDCs, to induce low levels of Tnf and Ccl3 in pDCs. It may also lead to the generation of an interferogenic synapse (❷) promoting local targeted delivery of viral material from the infected cell to pDCs, as illustrated on the upper left detailed drawing enlarged from the corresponding delimited area in the main drawing. This viral material is engulfed in pDCs and routed into dedicated endosomes, allowing TLR9 triggering, with the downstream enforcement of Tnf and Ccl3 expression and the induction of IFN-I genes. At this early activation state, pDCs from Ifnb1Eyfp reporter mice already start to express IFN-I but not yet clearly detectable levels of YFP. Then, pDCs further enhance their expression of IFN-I, leading to their expression of high levels of YFP in Ifnb1Eyfp reporter mice, and they simultaneously start to express IL-12 (❸). After termination of their IFN-I production, pDCs further enhance their IL-12 production, acquire CCR7 expression and migrate from the marginal zone to the white pulp through bridging channels (❹). Ultimately, pDCs relocate to the T cell zone where they harbor clear features of mature DCs, with a transcriptional, morphologic and functional convergence with tDCs, presumably including the acquisition of a dendritic morphology upon expression of Fscn1 and other genes involved into cytoskeleton remodeling, and acquisition of the ability to prime naïve CD4+ T cells (❺). CD169+ MMM, marginal zone metallophilic macrophages; mTNF, plasma membrane-bound TNF; pIRF7, phosphorylated IRF7.

Supplementary information

Supplementary Information

Supplementary Tables 1–3; Supplementary text corresponding to the sequence of the Eyfp gene in FASTA format.

Reporting summary

Supplementary Data 1

Pipeline and parameters used for the computational analysis of scRNA-seq data.

Supplementary Data 2

Source and gene content of the gene sets used for GSEA.

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Abbas, A., Vu Manh, TP., Valente, M. et al. The activation trajectory of plasmacytoid dendritic cells in vivo during a viral infection. Nat Immunol 21, 983–997 (2020).

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