Fig. 2: cDC proliferation is induced upon tissue entry and decreases upon CCR7 upregulation.

A Heatmaps representing relative expression of selected proliferation associated genes. B Proposed cDC differentiation pathway represented by RNA velocity vectors projected on the UMAP from Fig. 1B using scvelo. C Left: Representative flow cytometry plot showing the frequency of EdU⁺ cells amongst total SI LP cDCs, 24 h after i.p. injection of 1 mg EdU. cDCs were pre-gated as live, leucocytes, single cells, CD11c⁺MHCII⁺, CD64⁻. Right: Frequency of EdU⁺ cells amongst total cDCs, cDC subsets and macrophages (MΦ) 24 h after i.p. injection of 1 mg EdU. Data are presented as mean ± SD with each dot representing an individual biological replicate (n = 3). Statistical comparison was performed using a two-tailed Student´s t-test. D, E CCR7^gfp/+ mice were administered 1 mg BrdU by i.p. injection. BrdU incorporation was analysed 24, 48 or 72 h later amongst total SI LP cDCs (D) or amongst CCR7⁻ and CCR7⁺ SI LP cDCs (E) Data are presented as mean ± SD with each dot representing an individual biological replicate (n = 5 for 24 h from two experiments, n = 3 for 48 h and 72 h from one experiment) E The FACS plots show example staining at 24 h. The chart on the right shows the percentage of BrdU⁺ cells among CCR7⁻ (grey) and CCR7⁺ (black) SI LP cDCs. Statistical comparison was performed using a two-way ANOVA with Šídák’s multiple comparisons test. F Schematic illustration of the experimental setup. Generated in part using images adapted from Servier Medical Art (https://smart.servier.com/), licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). G Gating strategy and quantification of EdU incorporation amongst Dendra red⁻ (Dred⁻) and Dendra red⁺ (Dred⁺) cDCs and macrophages (MΦ). Cells were gated as live leucocytes, single cells, CD11c⁺MHCII⁺, and either as CD64⁺ macrophages or CD64⁻ cDCs. cDC subsets were further identified by differential expressions of XCR1 (cDC1s) and CD11b (cDC2s). n = 5, each dot represents an individual biological replicate, calculated as the mean percentage of EdU⁺ cells in the same population from two different segments of the same intestine. Data are presented as mean ± SD. Statistical comparison was performed using a two-tailed Student´s t-test. H Flow cytometry plots showing SI LP cDCs (live, single cells, CD45⁺, CD11c⁺MHCII⁺, CD64⁻) stained with either XCR1 (left) or with XCR1 and TLR3 (right). I Representative immunofluorescence images (left) and magnified areas of interest (right) of proximal SI sections stained with DAPI (grey) and anti-TLR3 (red) before (top) and after (bottom) applying Delaunay clustering with a 40 µm threshold. Colour code represents the number of cDC1s per cluster (cluster size). Scale bar lengths are indicated in the images. J Quantification of cDC1 (TLR3⁺) clusters and the number of cDC1s per cluster size (35 SI sections from 3 WT mice). K Example histogram and quantification of the mean fluorescent intensity (MFI) of surface MHCII amongst Dred⁺ and Dred⁻ SI LP cDCs 24 h after photoconversion. Data are presented as mean ± SD with each dot representing an individual biological replicate (n = 4). Statistical comparison was performed using a two-tailed Student´s t-test. L Gating strategy used for binning of the SI LP cDC population according to the surface MHCII expression ( ~ 25% of the CCR7⁻ cDC population per bin M1-M4) and CCR7 expression. M, N Quantification of the frequency of EdU⁺ cells, 2 h after i.p. injection of EdU, in bins representing cDC maturation (as shown in L) for total cDCs, cDC1s and cDC2s in WT and CCR7^gfp/+ (M) or CCR7^gfp/gfp mice (N). Data are presented as mean ± SD with each dot representing the mean of 7 (M) or 4 (N) biological replicates. Statistical comparison was performed using an ordinary one-way ANOVA. In all panels, asterisks indicate statistical significance (ns= not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). Source data and exact P-values are provided in the Source Data file.