Abstract
High-dimensional approaches have revealed heterogeneity amongst dendritic cells (DCs), including a population of transitional DCs (tDCs) in mice and humans. However, the origin and relationship of tDCs to other DC subsets has been unclear. Here we show that tDCs are distinct from other well-characterized DCs and conventional DC precursors (pre-cDCs). We demonstrate that tDCs originate from bone marrow progenitors shared with plasmacytoid DCs (pDCs). In the periphery, tDCs contribute to the pool of ESAM+ type 2 DCs (DC2s), and these DC2s have pDC-related developmental features. Different from pre-cDCs, tDCs have less turnover, capture antigen, respond to stimuli and activate antigen-specific naïve T cells, all characteristics of differentiated DCs. Different from pDCs, viral sensing by tDCs results in IL-1β secretion and fatal immune pathology in a murine coronavirus model. Our findings suggest that tDCs are a distinct pDC-related subset with a DC2 differentiation potential and unique proinflammatory function during viral infections.
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Data availability
Mass cytometry data generated in this study are deposited in FlowRepository (FR-FCM-Z6CW, FR-FCM-Z6CN and FR-FCM-Z6CK). RNA-seq data generated in this study are deposited in NCBI GEO under the accession codes GSE198553 and GSE198720. NanoString data are deposited in NCBI GEO under the accession code GSE233143. Source data are provided with this paper.
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Acknowledgements
This work was supported by NIH grants to J.I. (grant nos. AI158808 and AI163775), B.R. (grant nos. AI072571, AI147501 and AI128949) and O.A.P. (grant no. CA232666). F.B.S. and R.A.H. are recipients of the Dean’s Fellowship (Stanford), R.A.M.-A. is supported by a fellowship from SECTEI (CDMX, Mexico), H.M.R.-M. by an NSF GRFP fellowship (grant no. 2019276237), N.M.A. by the Damon Runyon postdoctoral fellowship (grant no. DRG 2408-20) and A.G.-B. by a KL2 Mentored Career Development Award (grant no. KL2TR003143). CyTOF was performed on the Stanford FACS Facility instrument funded by grant no. S10OD016318-01. We thank the Idoyaga and Reizis Lab members for technical support and discussions.
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Contributions
F.B.S., R.A.M.-A. and M.A.-H. contributed equally to this work by designing experiments, performing research, analyzing and interpreting the data, and writing the paper. The specific contributions of the authors were as follows. F.B.S., R.A.M.-A., M.A.-H. and J.I. conceptualized the study. F.B.S., R.A.M.-A., M.A.-H., O.A.P., B.R. and J.I. were responsible for the methodology. F.B.S., R.A.M.-A., M.A.-H., S.S., D.S., R.A.H., H.M.R.-M. and E.E. performed the formal analyses. F.B.S., R.A.M.-A., M.A.-H., S.S., T.J.Y., R.A.H., Z.R.L., R.A.L. and A.H.R. performed investigations. O.A.P., N.M.A. and A.D. were responsible for resources. F.B.S., R.A.M.-A., M.A.-H., S.S., D.S., R.A.H., H.M.R.-M. and A.G.-B. performed visualizations. F.B.S., R.A.M.-A., M.A.-H., A.G.-B., B.R. and J.I. wrote the paper. B.R. and J.I. were responsible for funding acquisition. J.I. supervised the project. J.I. was responsible for project administration.
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J.I. serves on the scientific advisory board of Immunitas Therapeutics. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 tDC are distinct transcriptionally.
a CD135-enriched splenocytes from hCD2EYFP mice were stained and sorted. Briefly, lineage containing CD3+/CD19+/NK1.1+/Ly6G+ cells were removed, and cells were gated using the strategy described in Fig. 1f. DC2 were further separated as EYFP+ and EYFP- cells. Shown are cells before (upper panels) and after sort (bottom panels). b MA plots of bulk-RNAseq data comparing tDC vs DC2 and tDC vs pDC. Genes with 2-fold Log2 change are shown in red (up) and blue (down) (see also Supplementary Table 4). c MA plot comparing tDC vs all other DC (pDC, DC2 and DC1). Genes with 2-fold Log2 change are shown in red (up) and blue (down) (see also Supplementary Table 1). d scRNAseq of splenic DC subsets. Splenic DCs were sorted as shown in Fig. 1c and sequenced using droplet-based genomics. After filtering, 2,110 cells were analyzed. UMAP of clusters detected using Seurat’s pipeline (right). Heatmap of top DEG between clusters (left). e DC signature scores generated using CIBERSORTx in the bulk-RNAseq data from (a) was overlayed on UMAP from (d). f As in (e), but DC signature scores were overlayed on the KNetL Plot from Fig. 1d.
Extended Data Fig. 2 tDC originate from BM progenitors.
a Gating strategy used for the analysis of skin-draining lymph node (LN) and lung DC subsets excluded. Lineage contains CD3+/CD19+/NK1.1+/Ly6G+ cells. b Bar graphs (mean + SD) showing the contribution of donor cells to LN and lung DC populations (gated as in (a)) in mixed BMC generated by transplanting 50% CD45.1 WT and 50% CD45.2 TCF4cKO BM, normalized to NK cells (n = 6 mice in 2 experiments). Statistics determined by two-way ANOVA with Dunnett’s multiple comparison test. c Bar graph (mean + SD) showing the percentage of splenic DC (gated as in Fig. 1f) labeled with the lineage tracing CD300cTdT (n = 4 mice in 3 experiments). d Adoptive cell transfer of 30,000 BM (left) or splenic (right) CX3CR1EGFP CD45.1 pDC into CD45.2 WT non-irradiated congenic mice. Recipient mice were euthanized at different time points (2–8-days) and the number of recovered cells (gated on CD45.1+ and CX3CR1+) calculated per 1000 cells transferred (mean + SD). N = 3/time point except n = 2 for splenic pDC at 8-days. N represents number of mice and experiments. e CyTOF analysis of blood and spleen DC, analyzed by UMAP. Cells were gated as CD3-CD19-NK1.1-Ly6G-. Populations were delineated manually (top) and colored by marker expression (bottom)(n = 2 mice in 2 experiments). f Spleen, blood and BM of hCD2EYFP mice were analyzed for the presence of EYFP+ cells, that were further separated in Ly6D+ pDC and CX3CR1+ tDC. Lineage includes CD3+/CD19+/NK1.1+/Ly6G+ cells. One representative of 3 experiments.
Extended Data Fig. 3 tDC originate from pro-pDC.
a CD135+ BM cells were enriched, sorted as CD45+/CD3-/CD19-/Ly6G−, and prepared for scRNA-seq. Cells with a pDC-specific gene enrichment score of >0.15 were selected and re-clustered using Seurat as shown in Fig. 3d. Violin plots show the expression of the indicated genes in the clusters defined in Fig. 3d. b As in (a) but GSEA of selected pathways from clusters defined in Fig. 3d. Bubble size indicates the normalized enriched score (NES), and color scale depicts False Discovery Rate (FDR). c TdTomato expression in bone marrow progenitor cells from CD300cTdT mice, gated as in Fig. 3j. One representative experiment. d Imm pDC, pro-pDC and pro-cDC from the BM of CX3CR1EGFP or hCD2EYFP CD45.1 mice were purified as described in Fig. 3j and transferred into CD45.2 WT mice. 2- and 4-days post-transfer, recipient mice were analyzed for transferred cells in the spleen. Shown is the number of cells recovered in the spleen of recipient mice (mean + SD). Imm pDC: n = 3 at 2- and 4-days. Pro-pDC: n = 3 at 2-days, and n = 5 at 4-days. Pro-cDC: n = 3 at 2-days, and n = 5 at 4-days. N represents number of mice and independent experiments. e BM cells from CX3CR1EGFP or hCD2EYFP CD45.1 mice were CD135-enriched and purified by cell sorting using the gating strategy shown in Fig. 3j. Purified progenitors were adoptively transferred into congenic non-irradiated CD45.2 WT mice and analyzed 4-days later in the spleen of recipient mice. Left panels show post-sort purity of progenitors. Right panels show transferred cells recovered in the spleen of recipient mice, analyzed using the gating strategy described in Fig. 1f. In both cases, lineage includes CD3+/CD19+/CD335+/Ly6G+ cells. One representative of ≥ 3 experiments.
Extended Data Fig. 4 tDC originate from DN progenitors.
a Gating strategy of CD135-enriched BM progenitors from hCD2EYFP mice. Cells were stained for flow cytometry. Lineage contain CD3+/CD19+/NK1.1+/Ly6G+ cells. EYFP labeling is shown in the lower panels. One representative of 3 experiments. b As in (a), but CD115 progenitors were further gated based on the expression Ly6C, whereas CD127 and DN progenitors were gated based on the expression of Ly6D and SiglecH (top). Bottom histograms show EYFP expression in each population. One representative of 3 experiments. c The schematic shows adoptive transfer of 15,000–30,000 from CX3CR1EGFP CD45.1 BM progenitors into WT CD45.2 non-irradiated congenic mice. Transferred cells were identified in the spleen of recipient mice as CD45.1+ and CX3CR1+ cells. Bar graphs (mean + SD) show the percentage of each population recovered in the spleen (gated as shown in Fig. 1f). N ≥ 2/mice and experiments. d As in (c), but bar graphs (mean + SD) number of cells recovered in the spleen of recipient mice. N represents number of mice and independent experiments.
Extended Data Fig. 5 tDC convert into DC2.
a tDC were purified using the gating strategy described in Fig. 1f. Top graph shows cell purity after sort. Bottom graphs show the outcome of transferred cells in the spleen of recipient mice analyzed at 4-days. One representative of 6 experiments. b Bar graphs (mean + SD) of the total number of splenic DC in hCD2-iCre+/− Cx3cr1DTR+/− mice inoculated with DT and analyzed 1 day after (n = 5 mice in 4 experiments), or inoculated with DT every second day and analyzed 7 days later (n = 5 mice in 4 experiments). ‘C’ represents control mice (n = 8 mice in 4 experiments), which are a combination of hCD2-iCre−/− Cx3cr1DTR+/− mice inoculated with DT or hCD2-iCre+/− Cx3cr1DTR+/− mice inoculated with PBS (no differences were observed between these control mice). Statistics determined by one-way ANOVA with Tukey’s multiple comparison test. c As in (b), but BM progenitors from hCD2EYFP CX3CR1DTR mice were analyzed after 7 days of DT inoculation (n = 4 mice). Control mice are hCD2EYFP CX3CR1DTR mice inoculated with PBS (n = 3 mice). Bar graphs (mean + SD) of the frequency of total progenitor populations from Fig. 3j. Data pooled from 2 experiments. Statistical differences were determined by unpaired two-tailed t-test. d Mouse splenic DC subsets were sorted using the gating strategy show in Figs. 1f and 4g, and analyzed by PCR assay for IgH D-J rearrangements. Actin and IgH germline (GL) are also shown. One representative of 3 experiments. e DC2 from CD300cTdT mice were gated as indicated in Fig. 4f, and further separated in TdTomato+ and TdTomato-. Heatmap indicating the relative expression (Z-score of gMFI) of surface markers. Z-score was calculated base of n = 2 mice in 2 experiments.
Extended Data Fig. 6 tDC have lower turnover rate than pre-DC2 and required IRF4.
a Mice were inoculated i.p. with 1 mg of BrdU on day 0 and then fed continuously for 14 days with 0.8 mg/mL of BrdU in drinking water. After 14 days, BrDU was removed. Mice were euthanized at different time points post-BrdU removal, spleen cell suspension prepared and stained for BrdU and the identification of DC subsets as described in Figs. 1f and 5e. N ≥ 2/mice per time point in 2 independent experiments. b Expression of Ki-67 in each splenic DC population gated as described in Fig. 1f and 5e, analyzed by flow cytometry. One representative of 3 experiments. c BM cells from IRF4cKO or IRF4control mice were cultured with FLT3L as described in Fig. 2c. At day 6, tDC were sorted and re-cultured in complete media and 1% FLT3L for 1 and 2 days. Bar graphs (mean + SD) show the percentage of each DC recovered. N = 3 biologically independent samples in 3 experiment. d Bar graphs (mean + SD) of the total number of splenic DC (gated a Fig. 1f and 4f) in hCD2EYFP CX3CR1DTR mice inoculated with DT every second day (n = 3 mice) or left untreated (n = 3 mice), and analyzed at day 10. Statistics determined by unpaired two-tailed t-test. e As in (d), but shown is the number of other immune cells.
Extended Data Fig. 7 tDC respond to microbial stimulation and uptake antigen in vivo.
a Splenic DC subsets purified as described in Figs. 1f and 4f were analyzed by Nanostring. Heatmap shows the expression of TLRs by each DC subset. N = 2 mice in 2 experiments. b Whole spleen cell suspensions were activated with LPS, MPLA, Resiquimod or PolyIC for 6 hrs. Heatmap showing the upregulation of MHCII and CD86 in pDC, tDC, ESAM+ EYFP+ and EYFP- DC2, and DC1. Min and max correspond to the lowest and highest gMFI per row, respectively. N = 6 mice per group. Data pooled from 4 experiments. c As in (b), but bar graphs (mean + SD) of cytokine production. Whole spleen cell suspensions were activated with LPS (n = 6), MPLA (n = 4), Resiquimod (n = 6) or PolyIC (n = 6) for 6 hrs. TNF and IL-12p40 were detected by intracellular cytokine staining. N represents mice and data was pooled from 4 experiments. Statistical differences were determined by One-way ANOVA with Tukey’s multiple comparisons test. d Mice were inoculated with yellow-green polystyrene (YG-PS) beads i.v. Three hrs later, spleens were harvested and the phagocytosis of the particle was assessed by flow cytometry in each DC population, gated as in Fig. 1f (n = 3 mice in 3 experiments). Bars represent mean + SD. Statistical differences were determined by One-way ANOVA with Tukey’s multiple comparisons test. e As in (d), but mice were inoculated with PKH26-labeled SRBC (n = 6 mice in 4 experiments).
Extended Data Fig. 8 Number of immune cells, viral titer and ALT in pDC- and pDC/tDC- depleted animals infected with M-CoV.
a Liver cell suspensions were enriched in immune cells using a Percoll gradient, and stained with a cocktail of Ab for the analysis of neutrophils and lymphocytes. One representative of 3 experiments. b As in (b), but cell suspensions were stained for the analysis of DC and other myeloid cells. Lineage contains CD3+/CD19+/NK1.1+/Ly6G+ cells. One representative of 3 experiments. c Liver of WT mice were analyzed for the number of DC subsets by flow cytometry 2- and 5-days post-M-CoV infection. Fold change of the number of each subset to day 0 is shown. N = 3 at 0 days, n = 14 at 2-days, and n = 11 at 5-days. Data pooled from ≥ 4 experiments. p-values represent statistical differences between tDC and DC2 vs DC1. d Liver immune cell numbers 2-days post-M-CoV infection of pDCΔ (n = 3); pDCΔtDCΔ (n = 6) and control (n = 5) mice. pDCΔ mice are BDCA2-DTR+/− mice inoculated with DT one day before M-CoV infection. pDCΔtDCΔ are hCD2EYFP CX3CR1DTR mice inoculated with DT every other day for 5-days before M-CoV infection. Control mice are a combination of BDCA2-DTR+/− or hCD2EYFP CX3CR1DTR mice inoculated with PBS, and DTR−/− mice inoculated with DT (no difference in control mice was observed). Shown is mean + SD. N represents independent mice in 2 experiments. e As in (d), but serum ALT values (mean + SD) were determined 2-, 5- and 7-days post-M-CoV infection. N ≥ 4 mice/group/time point in 4 experiments. f As in (e), but viral titers (log10 pfu/gr. Tissue) in liver (top) and spleen (bottom) were determined 2-, 5- and 7-days post-M-CoV infection. N ≥ 4 mice/group/time point. Violin plots show distribution of data pooled from 4 exp. Statistical differences determined by Two-way ANOVA with Tukey’s multiple comparisons test (c,e,f), or One-way ANOVA with Tukey’s multiple comparisons test per subset (d).
Extended Data Fig. 9 tDC promote immune pathology following M-CoV infection.
a TCF4cKO vs TCF4control mice were generated by transplanting Itgax-Cre+/− Tcf4fl/fl or Itgax-Cre−/− Tcf4fl/fl BM into lethally irradiated CD45.1 WT mice, respectively. 2 months post-reconstitution, mice were infected with M-CoV, and the frequency of liver DC (mean + SD) analyzed 2-days later (n = 6 mice/group in 2 experiments). b Percentage weight loss (left, mean + SD) and survival (right) of TCF4cKO (n = 7) vs TCF4control (n = 10) mice in 3 experiments. c As in (b), but viral titers (log10 pfu/gr. tissue) were determined 5-days post-M-CoV infection. N = 7 mice for TCF4control and n = 6 mice for TCF4cKO. Violin plots show distribution from 3 experiments. d As in (b), but shown is serum ALT (mean + SD; n = 6 mice/group in 3 experiments). e Serum cytokines were determined by Luminex in control (n = 7 mice), pDCΔ (n = 8) and pDCΔtDCΔ (n = 8) 2-days p.i. in 4 experiments. Shown are cytokines different between pDCΔ vs pDCΔtDCΔ mice. f Percentage weight loss (left, mean + SD) and survival (right) of pDCΔ mice inoculated with anti-NK1.1 Ab or isotype control on day −1, 0 and 2 post-infection with M-CoV. N = 3 mice/group except n = 5 mice for pDCΔ + isotype, pooled from 2 experiments. g As in (f), but viral titers were evaluated at 5-days p.i. (n = 3 mice/group). Violin plots show distribution from 2 experiments. h As in Fig. 8j, but splenic viral titers (log10 pfu/gr. tissue) were determined. N = 5 mice/group except n = 8 for pDCΔ + control Ab. Violin plots show distribution from 2 experiments. i As in Fig. 8k but heatmaps show secretion of TNF, IL-6 and IL-12p70 by DC subsets (N = 4 mice/subset in 4 experiments). Statistical differences were determined by unpaired two-tailed t-test (a,c-d), One-way ANOVA (e,h) or Two-way ANOVA (b,f) with Tukey’s multiple comparisons test, and Mantel-Cox test for survival (b,f).
Extended Data Fig. 10 Summary of DC development at steady state.
Diagram of the DC network incorporating tDC development.
Supplementary information
Supplementary Information
Uncropped and unprocessed gels for Extended Data Fig. 5d.
Supplementary Tables
Supplementary Table 1: Differentially expressed genes between tDC and all other DC subsets. Significance threshold set to Padj < 0.05. Supplementary Table 2: Differentially expressed genes between clusters of KNetL graph. Calculated using findMarkers function (padjval < 0.05 and fold.change > 2). Supplementary Table 3: List of genes shown in Fig. 1e were grouped by KNetL clusters and annotated if significant in pair-wise comparisons of tDC versus pDC and tDC versus DC2 of bulk RNA-seq data (threshold set to Padj < 0.05). Supplementary Table 4: Differentially expressed genes between tDC versus pDC, and tDC versus DC2. Significance threshold set to Padj < 0.05. Supplementary Table 5: Differentially expressed genes between EYFP+ DC2 and EYFP− DC2. Significance threshold set to Padj < 0.05.
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Sulczewski, F.B., Maqueda-Alfaro, R.A., Alcántara-Hernández, M. et al. Transitional dendritic cells are distinct from conventional DC2 precursors and mediate proinflammatory antiviral responses. Nat Immunol 24, 1265–1280 (2023). https://doi.org/10.1038/s41590-023-01545-7
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DOI: https://doi.org/10.1038/s41590-023-01545-7
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