Research ArticleINNATE IMMUNITY

Tissue clonality of dendritic cell subsets and emergency DCpoiesis revealed by multicolor fate mapping of DC progenitors

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Science Immunology  01 Mar 2019:
Vol. 4, Issue 33, eaaw1941
DOI: 10.1126/sciimmunol.aaw1941
  • Fig. 1 Pre-cDCs and cDCs actively cycle in lymphoid and nonlymphoid organs.

    (A) DNA content histogram (top) or dot plot with DNA content and phospho-H3 (bottom) of BM CDPs from one mouse as a representative example of cell cycle analysis. (B, D, F, H, and J) Percentage of cells in G0/G1, S, G2, or M phases of the cell cycle determined as in (A) in the BM (B), spleen (D), mesLN (F), SI (H), and lung (J). (C, E, G, I, and K) Percentage of Ki67+ cells in the BM (C), spleen (E), mesLN (G), SI (I), and lung (K). Data in (B, D, F, H, and J) are mean values from six C57BL/6 mice. Data in (C), (E), (G), (I), and (K) are compiled from 12 C57BL/6 mice analyzed in two independent experiments. Error bars correspond to variation across mice using SD. Cells are gated as indicated in Materials and Methods.

  • Fig. 2 Spectral imaging of organs from Clec9aConfettireveals the presence of single-color cDC clusters.

    (A) Workflow of tissue processing, staining, and imaging of Clec9aConfetti mouse organs. (B) 3D projection of a 300-μm spleen section stained with anti-CD169. Confetti surfaces were generated with Imaris software to reduce autofluorescence. Zooming into T cell areas (1) was used to visualize Confetti+ cDCs (2). (C) 3D projection of a mesLN with surfaces as in (B). Square depicts selected zoom in area displayed at the bottom. (D) 3D projection of a 300-μm vibratome section of the SI from a Clec9aConfetti mouse stained for E-cadherin to delineate the epithelium. (E) 3D projection of a 300-μm vibratome section of the lung from a Clec9aConfetti mouse. Autofluorescence channel is displayed to visualize the lung structure.

  • Fig. 3 Analysis of single-color cDC clusters using ClusterQuant 3D.

    (A) Workflow of plane separation (1), manual cell annotation (2), 3D Voronoi generation, Monte Carlo (MC) randomization (4), and analysis using ClusterQuant 3D. (B) Original images from the SI (top) and the lung (bottom) were annotated using ClusterQuant 3D software and converted to Voronoi polyhedrons (middle), which were then randomized through a Monte Carlo simulation (right). CD64 staining was used to exclude CD64+ cells from the analysis. Dashed lines indicate the structure of the SI (villi) or lung (airways), determined from CD11b staining or autofluorescence channel, respectively. Colors represent the different Confetti fluorescent proteins. Note the scarcity of double-labeled cells, especially in lung.

  • Fig. 4 SI and lung cDCs are organized in spatially restricted clusters of sister cells.

    (A) From left to right: Number of clusters of two or more cells normalized to a number of cells, percentage of number of cells in clusters, and cluster compactness from 24 images of SI from four mice. Each point represents one image; observed (O, orange) compared with simulations (S, gray). (B) As in (A) from 25 lung images from five mice. (C) Data from (A) grouped per mouse. Lines link the observed and simulated scenarios associated with each mouse. Colors correspond to individual mice (observed and simulation). (D) Data from (B) grouped per mouse as for (C). (E and F) Proportion of clusters of the indicated size in observed scenario (orange) normalized to the simulated scenario (dashed lines). Data are from SI (E) or lung (F) of a representative mouse. Other mice are shown in fig. S4. (G) Proportion of all clusters analyzed of size 1 (gray) or ≥2 in the SI (orange) or lung (blue). (H) Comparison of proportion of clusters of size 2 to 10 in the SI (orange) and in the lung (blue). In all cases, CD64 staining was used to exclude CD64+ cells from the analysis. Statistical analysis in (B) to (E) used a paired t test, and in (F) and (G), used a χ2 test.

  • Fig. 5 Single-color cDC clusters are predominantly composed of a single cDC subset.

    (A) 3D projection of a Clec9Confetti image of the SI (left) or lung (right) stained for CD103, CD11b, and CD64. (B and C) Single z optical slices depicting individual cells from the SI (B) or lung (C) of Clec9Confetti mice; individual channels are shown on the right side of the merged image. (D) Pie charts representing percentage of pure clusters in the SI (orange, 80%) or lung (blue, 70%) were analyzed. Gray bar indicates mixed clusters. Data are pooled from all images, and n indicates the number of clusters analyzed. (E and F) Analysis of the proportion of pure clusters of cluster size 2 to 7 found in the SI (E, orange) or lung (F, blue) (o) versus the expected null distribution assuming random mixing (r, gray). Data are pooled from all images. Error bars correspond to variation across mice using SD. Statistical analysis was carried out using a Fisher’s exact test.

  • Fig. 6 Influenza A virus infection dilutes single-color cDC clustering.

    (A) Number of cDC1s (left) or cDC2s (right) in the lungs of Clec9aeYFP mice infected with influenza A virus (magenta) or non-infected (N.i., gray). (B) 3D projection of a Clec9Confetti lung section 7 dpi with influenza A virus. (C) Quantification of Confetti+ cells, CD64+ cells, and X31 particles from images with high (magenta) and low (purple) infiltration of cells. (D) Number of clusters of two or more cells normalized to number of cells and percentage of cells in clusters from 20 high infiltrated lung images from five mice. Each point represents one image; observed (magenta) compared with simulations (gray). (E) Data in (D) grouped per mouse. Lines link the observed and simulated scenarios associated with each mouse. Colors correspond to individual mice (observed and simulations). (F) Data as in (D) from 19 low infiltrated lung images from four mice. (G) Data in (F) grouped per mouse. (H and I) Proportion of clusters of the indicated size in original (O) normalized to the simulated (S, dashed lines) scenario from the high (H) or low (I) infiltrated areas of a representative mouse. (J) Proportion of clusters of size 1 (gray) or ≥2 in non-infected lungs (blue) versus. high (magenta) and low (purple) infiltrated areas from lungs of infected mice. n.s., not significant. (K) Comparison of proportion of clusters of size 2 to 10 in non-infected lungs (blue; Fig. 2) and high (magenta) and low (purple) infiltrated areas in the lungs of infected mice. Data correspond to the pool of all mice analyzed. Statistical analysis used an unpaired t test (A) in two independent experiments with three mice per group, paired t test (C to G), and χ2 test (H to K).

  • Fig. 7 Influenza A virus infection increases lung cDC numbers by recruiting BM progenitors.

    (A and B) Percentage of the lung pre-cDCs and cDCs (A) and of the BM CDPs and pre-cDCs (B) in G0/G1, S, G2, or M phases of the cell cycle determined as in Fig 1. Data are mean values from six non-infected (top) or six C57BL/6 mice 7 dpi with influenza virus (bottom). (C to F) Numbers of BM CDPs (D) or pre-cDCs in the lung (C), BM (E), or blood (F) in non-infected (gray), or influenza A virus–infected C57BL/6 mice (magenta) at 3 or 7 dpi. Each dot represents one mouse of six per group from one representative experiment. (G) Relative mean percentage of cells from peripheral blood of 41 patients before and after natural infection with influenza A virus obtained from microarray data using CIBERSORT. Tregs, regulatory T cells; NK cells, natural killer cells. (H) Percentage of activated blood DCs in individual patients from (G). (I) Expression of SEMA4D, CD3E, CD79A, and CD79B in peripheral blood from patients before (pre, gray) or in the first 48 hours of symptoms after natural infection with influenza A virus (post, magenta). Statistical analysis in (C) to (F) was based on an unpaired t test in a single experiment with six mice per group. Statistics in (H) and (I) used a paired t test.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/33/eaaw1941/DC1

    Supplementary Methods

    Fig. S1. Staining for Ki67 in cDCs and their progenitors.

    Fig. S2. Flow cytometric analysis of Clec9aConfetti organs.

    Fig. S3. Analysis of Clec9aCre-based fate-mapping mice on a Flt3l−/− background.

    Fig. S4. ClusterQuant 3D analysis of single-color cDC clustering in Clec9aConfetti mice.

    Fig. S5. Analysis of Confetti+ cDC subsets.

    Fig. S6. Quantification of infected cells and cell infiltration during influenza A virus infection.

    Fig. S7. Voronoi diagrams of analyzed high and low infiltrated areas from infected Clec9aConfetti mice.

    Fig. S8. Cluster size distribution in high and low infiltrated areas of lungs from infected Clec9aConfetti mice.

    Fig. S9. Quantification of cDC proliferation and pre-cDC composition during influenza A virus infection.

    Fig. S10. Flow cytometry gating strategy for CDPs.

    Fig. S11. Flow cytometry gating strategy for cDC subsets and macrophages.

    Fig. S12. Flow cytometry gating strategy for Confetti+ cells in Clec9aConfetti mice.

    Table S1. Excel file with raw data used to generate all graphs that have n < 25.

    Table S2. Abs used in flow cytometry.

    Table S3. Abs used in confocal microscopy.

    Movie S1. SI from a Clec9aConfetti mouse.

    Movie S2. Lung from a Clec9aConfetti mouse.

    Movie S3. SI from a Clec9aConfetti mouse crossed to a Flt3l−/− background.

    Movie S4. Lung from a Clec9aConfetti mouse crossed to a Flt3l−/− background.

    Movie S5. Representative annotated image of the SI of a Clec9aConfetti mouse.

    Movie S6. Representative annotated imaged of the lung of a Clec9aConfetti mouse.

    Movie S7. SI from a Clec9aConfetti mouse stained for cDC subsets.

    Movie S8. Lung from a Clec9aConfetti mouse stained for cDC subsets.

    Movie S9. Image an area of the lung of a Clec9aConfetti 1 dpi with influenza A virus and stained for viral proteins.

    Movie S10. Representative annotated imaged of a high infiltrated area of the lung of a Clec9aConfetti 7 dpi with influenza A virus.

    Movie S11. Representative annotated imaged of a low infiltrated area of the lung of a Clec9aConfetti 7 dpi with influenza A virus.

    References (47, 48)

  • Supplementary Materials

    The PDF file includes:

    • Supplementary Methods
    • Fig. S1. Staining for Ki67 in cDCs and their progenitors.
    • Fig. S2. Flow cytometric analysis of Clec9aConfetti organs.
    • Fig. S3. Analysis of Clec9aCre-based fate-mapping mice on a Flt3l−/− background.
    • Fig. S4. ClusterQuant 3D analysis of single-color cDC clustering in Clec9aConfetti mice.
    • Fig. S5. Analysis of Confetti+ cDC subsets.
    • Fig. S6. Quantification of infected cells and cell infiltration during influenza A virus infection.
    • Fig. S7. Voronoi diagrams of analyzed high and low infiltrated areas from infected Clec9aConfetti mice.
    • Fig. S8. Cluster size distribution in high and low infiltrated areas of lungs from infected Clec9aConfetti mice.
    • Fig. S9. Quantification of cDC proliferation and pre-cDC composition during influenza A virus infection.
    • Fig. S10. Flow cytometry gating strategy for CDPs.
    • Fig. S11. Flow cytometry gating strategy for cDC subsets and macrophages.
    • Fig. S12. Flow cytometry gating strategy for Confetti+ cells in Clec9aConfetti mice.
    • Legend for table S1
    • Table S2. Abs used in flow cytometry.
    • Table S3. Abs used in confocal microscopy.
    • Legends for movies S1 to S11
    • References (47, 48)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Excel file with raw data used to generate all graphs that have n < 25.
    • Movie S1 (.avi format). SI from a Clec9aConfetti mouse.
    • Movie S2 (.avi format). Lung from a Clec9aConfetti mouse.
    • Movie S3 (.avi format). SI from a Clec9aConfetti mouse crossed to a Flt3l−/− background.
    • Movie S4 (.avi format). Lung from a Clec9aConfetti mouse crossed to a Flt3l−/− background.
    • Movie S5 (.avi format). Representative annotated image of the SI of a Clec9aConfetti mouse.
    • Movie S6 (.avi format). Representative annotated imaged of the lung of a Clec9aConfetti mouse.
    • Movie S7 (.avi format). SI from a Clec9aConfetti mouse stained for cDC subsets.
    • Movie S8 (.avi format). Lung from a Clec9aConfetti mouse stained for cDC subsets.
    • Movie S9 (.avi format). Image an area of the lung of a Clec9aConfetti 1 dpi with influenza A virus and stained for viral proteins.
    • Movie S10 (.avi format). Representative annotated imaged of a high infiltrated area of the lung of a Clec9aConfetti 7 dpi with influenza A virus.
    • Movie S11 (.avi format). Representative annotated imaged of a low infiltrated area of the lung of a Clec9aConfetti 7 dpi with influenza A virus.

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