Fig. 3: cDC maturation is accompanied by a gradual increase of costimulatory molecules.

A Proposed cDC1 differentiation pathway represented by RNA velocity vectors of the reclustered scRNA-seq data corresponding to clusters 7, 8, 0 and 9 from the UMAP in Fig. 1B. B Heatmap of the UMAP as shown in A with overlaid pseudotime values. C Violin plots showing the expression of selected migration-associated genes, cDC1 subset markers and co-stimulatory molecules on the cells of cDC1 clusters as defined in Fig. 3A. Numbers along the x-axes indicate the clusters identified in A and B, ordered according to the trajectories suggested by the RNA velocity and pseudotime analyses. D Flow cytometry analysis of SI LP cDC1 from CCR7^gfp/+ mice gated along maturation bins as in Fig. 2L. Histograms show example staining of the indicated markers. The graphs show the normalised mean fluorescent intensity (nMFI) of selected cDC1 subset markers and co-stimulatory molecules along cDC1 maturation states. Data are shown as mean ± SD of 7 biological replicates pooled from two independent experiments. E Proposed cDC2 differentiation pathway represented by RNA velocity vectors of the reclustered scRNA-seq data corresponding to clusters 2, 5, 3, 1, 10, 4, 6 and 9 from the UMAP in Fig. 1B. F Heatmap of the UMAP as shown in E with overlaid pseudotime values. G Violin plots showing the expression of selected migration-associated genes, cDC2 subset markers, and co-stimulatory molecules on the cells of cDC2 clusters as defined in Fig. 3E. Numbers along the x-axes indicate the clusters identified in E and F, ordered according to the trajectories suggested by the RNA velocity and pseudotime analyses. H, I Flow cytometry analysis of CD103⁻ (H) or CD103⁺ (I) SI LP cDC2s from CCR7^gfp/+ mice gated along maturation bins as in Fig. 2L. Histograms show example staining of the indicated markers. The graphs show the nMFI of selected cDC2 subset markers and co-stimulatory molecules along cDC2 maturation states. Data are shown as mean ± SD of 7 biological replicates pooled from two independent experiments. In panels D, H and I, the nMFI of the indicated marker was normalised relative to the MFI value of the M1 maturation bin which was set to 1. 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.