Molecular characterization of Ltbr-deficient FRCs in murine lymph nodes. (A–C) scRNA-seq data of FRCs and VSMCs from mesenteric lymph nodes of Ccl19-iEYFP and Ccl19-iEYFP Ltbr^fl/fl mice. (A) UMAP representation colored according to cluster identity. (B) Dot plot showing the average expression of signature genes in VSMCs and FRC subsets of EYFP⁺ cells isolated from mesenteric lymph nodes of Ccl19-iEYFP and Ccl19-iEYFP Ltbr^fl/fl mice. (C) Pie chart showing the relative abundance of FRC subsets and VSMCs in mesenteric lymph nodes from Ccl19-iEYFP and Ccl19-iEYFP Ltbr^fl/fl mice. (D and E) Confocal microscopy images showing cross sections of mesenteric lymph nodes from Ccl19-iEYFP Ltbr^fl/fl mice. Boxed areas indicate regions of higher magnification. Arrows indicate appearance of Ccl19-tTA⁺ cells in perivascular areas. Microscopy images are representative for three mesenteric lymph nodes from three independent experiments. Scale bars: 500 µm (D) and 30 µm (E). (F and G) Fate analysis of EYFP⁺ cells in mesenteric lymph nodes harvested from adult Ccl19-iEYFP⁺ (F) and Ccl19-iEYFP Ltbr^fl/fl (G) mice after Dox administration from E18. Microscopy images are representative for four mesenteric lymph nodes per condition from three independent experiments. Scale bars: 40 µm (F–G). (H and I) Quantification of perivascular Ccl19-iEYFP⁺ cells using histology of cross sections from mesenteric lymph nodes of fate mapped Ccl19-iEYFP and Ccl19-iEYFP Ltbr^fl/fl mice. (H) Average pixel intensity of Ccl19-iEYFP signal with distance from CD31⁺ blood vessels (BV). (I) Quantification of perivascular grey values of EYFP signal in Ccl19-iEYFP and Ccl19-iEYFP Ltbr^fl/fl mice. Data are representative of four mesenteric lymph nodes from three independent experiments (H and I). (J) Schematic depiction of differentiation trajectories of CCL19-expressing LTo cells in murine lymph nodes. Figure was complemented with elements from https://BioRender.com.