Journal: The Journal of Experimental Medicine
Article Title: CD301b + monocyte-derived dendritic cells mediate resistance to radiotherapy
doi: 10.1084/jem.20231717
Figure Lengend Snippet: RT induces accumulation of moDCs in the TME. (A and B) B16-bearing B6 mice on days 0 (−RT) and 5 after RT (+RT), analyzed by CyTOF. Mononuclear phagocytes were gated as live/singlets/CD45 + /CD3 − /CD19 − /CD335 − /CD8 − , and eosinophils/neutrophils were excluded ( n = 2/group, 2 experiments [exp.]). (A) Left: UMAP generated using FlowSOM. Right: UMAPs of all cells and treatments (contour plot) were overlaid with cell populations on days 0 (−RT) and 5 after RT (+RT). (B) Heatmap of the Z-scored expression of markers in each cluster identified by FlowSOM. (C and D) B16 tumors analyzed by flow cytometry. (C) Representative gating strategy of mononuclear phagocytes gated on live/singlets/CD45 + /Ly6G − /lineage − (lineage staining includes CD3, CD19, and NK1.1). (D) As in C, but the expression of F4/80, CD11c, and CD26 on cells within gate E is shown. (E) Representative expression of canonical markers, by flow cytometry. Numbers indicate gMFI ×10 2 . Myeloid DCs are shown on days 0 (−RT) and 5 after RT (+RT). All the rest of the populations are shown on day 5 after RT (1 of 3 exp.). (F) Cell numbers per mg of tumor on days 1, 3, and 5 after RT, by flow cytometry (mean + SD; n = 4–20/group, 4–5 exp.). Statistics: one-way ANOVA plus Tukey’s post hoc test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (G) Mononuclear phagocytes were analyzed on days 0 and 5 after RT, by scRNAseq. 5,000 mononuclear phagocytes (excluding granulocytes and pDCs) were plotted. (H) Heatmap displays the top five DEGs for each cluster. Gene expression levels are normalized as log-transformed counts per cell for each cluster, as indicated in . (I) Normalized gene expression levels for the indicated genes, by scRNAseq. (J) moDC signatures overlaid onto the UMAP of scRNAseq data. Expression values represent log-normalized counts for genes listed in . gMFI, geometric mean fluorescence intensity.
Article Snippet: CD45 + cells from tumor single-cell suspensions were enriched on days 0 and 5 after RT by positive selection using αCD45-Biotin and αBiotin magnetic microbeads (Miltenyi).
Techniques: Generated, Expressing, Flow Cytometry, Staining, Gene Expression, Transformation Assay, Fluorescence
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![RT induces accumulation of moDCs in the TME. (A and B) B16-bearing B6 mice on days 0 (−RT) and 5 after RT (+RT), analyzed by CyTOF. Mononuclear phagocytes were gated as live/singlets/CD45 + /CD3 − /CD19 − /CD335 − /CD8 − , and eosinophils/neutrophils were excluded ( n = 2/group, 2 experiments [exp.]). (A) Left: UMAP generated using FlowSOM. Right: UMAPs of all cells and treatments (contour plot) were overlaid with cell populations on days 0 (−RT) and 5 after RT (+RT). (B) Heatmap of the Z-scored expression of markers in each cluster identified by FlowSOM. (C and D) B16 tumors analyzed by flow cytometry. (C) Representative gating strategy of mononuclear phagocytes gated on live/singlets/CD45 + /Ly6G − /lineage − (lineage staining includes CD3, CD19, and NK1.1). (D) As in C, but the expression of F4/80, CD11c, and CD26 on cells within gate E is shown. (E) Representative expression of canonical markers, by flow cytometry. Numbers indicate gMFI ×10 2 . Myeloid DCs are shown on days 0 (−RT) and 5 after RT (+RT). All the rest of the populations are shown on day 5 after RT (1 of 3 exp.). (F) Cell numbers per mg of tumor on days 1, 3, and 5 after RT, by flow cytometry (mean + SD; n = 4–20/group, 4–5 exp.). Statistics: one-way ANOVA plus Tukey’s post hoc test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (G) Mononuclear phagocytes were analyzed on days 0 and 5 after RT, by scRNAseq. 5,000 mononuclear phagocytes (excluding granulocytes and pDCs) were plotted. (H) Heatmap displays the top five DEGs for each cluster. Gene expression levels are normalized as log-transformed counts per cell for each cluster, as indicated in . (I) Normalized gene expression levels for the indicated genes, by scRNAseq. (J) moDC signatures overlaid onto the UMAP of scRNAseq data. Expression values represent log-normalized counts for genes listed in . gMFI, geometric mean fluorescence intensity.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9126/pmc11949126/pmc11949126__jem_20231717_fig2.jpg)
![Myeloid cell analysis after RT. (A and B) B16 tumors analyzed by CyTOF. (A) Representative gating strategy of live/singlets/CD45 + /Ly6G − /lineage − (lineage staining includes CD3, CD19, and NK1.1). (B) Left: UMAP showing clusters generated using FlowSOM . Right: cell populations identified via CyTOF gating were overlaid onto the depicted UMAP. (C) Representative expression of canonical markers, by flow cytometry. Numbers indicate gMFI ×10 2 . Myeloid DCs are shown on days 0 (−RT) and 5 (+RT) after RT, while all the rest of the populations are shown at 5 days after RT ( n = 1 of 3 experiments [exp.]). (D) BRaf/Pten tumors analyzed on days 1, 3, and 5 after RT, by flow cytometry (mean + SD; n = 5–19/group, 3–5 exp.). Statistics: one-way ANOVA plus Tukey’s post hoc test (D). *P ≤ 0.05, **P ≤ 0.01. gMFI, geometric mean fluorescence intensity.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9126/pmc11949126/pmc11949126__jem_20231717_figs2.jpg)
![CD301b is preferentially expressed on moDCs after RT. (A and B) Mononuclear phagocytes in Ms4a3 Cre × Rosa LSL-TdTomato B16–bearing mice on days 0 and 5 after RT, analyzed by flow cytometry. (A) Left: representative histograms of TdTomato expression. Numbers represent the frequency of cells expressing TdTomato. Right: frequency of TdTomato + and TdTomato − cells in Ms4a3 Cre × Rosa LSL-TdTomato tumor–bearing mice (mean; n = 4/group, 2 experiments [exp.]). (B) Total number of TdTomato + and TdTomato − myeloid DCs in Ms4a3 Cre xRosa LSL-TdTomato tumor–bearing mice (mean + SD; n = 4/group, 2 exp). (C) Bone marrow CD45.2 Ccr2 RFP+ Cx3cr1 EGFP+ monocytes were adoptively transferred into B16-bearing CD45.1 mice 2 days after RT. Left: experimental design. Right: numbers and identity of transferred cells 5 days after RT, by flow cytometry (mean + SD; n = 3–4/group, 2 exp.). (D) Expression of Ki-67 by mononuclear phagocytes on days 0 and 5 after RT, by flow cytometry. (E) Top: relative expression of Cd301b is overlaid onto the UMAP from , by scRNAseq; expression values represent log-normalized counts. Bottom: relative expression of CD301b is displayed onto the UMAP of all populations on days 0 and 5 after RT , by CyTOF; expression values represent an arcsinh transformation of marker intensity. (F) Relative expression of CD301b in B16 tumors on days 0 and 5 after RT, by flow cytometry ( n = 1 of 2 exp.). Numbers represent the frequency of positive cells. (G) Number of CD301b + myeloid DCs per mg of tumors in B16 (left), and BRaf/PTEN (right) on days 0 and 5 after RT, by flow cytometry (mean + SD; n = 7–9/group, 2–3 exp.). (H) Left: expression of CD301b and Ms4a3 Cre × Rosa LSL-TdTomato on days 0 and 5 after RT, by flow cytometry. Right: number of TdTomato + CD301b + myeloid DCs per mg of tumor on days 0 and 5 after RT, by flow cytometry (mean + SD; n = 4/group, 2 exp.). Statistics: two-tailed t test (A–C, G, and H). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9126/pmc11949126/pmc11949126__jem_20231717_fig3.jpg)