Characterization of Ltbr-deficient FRCs in peripheral lymph nodes. (A–C) scRNA-seq analysis of FRCs and VSMCs from peripheral lymph nodes of Ccl19-iEYFP and Ccl19-iEYFP Ltbr^fl/fl mice. (A) UMAP representation colored by cluster identity. (B) Dot plot showing the average expression of signature genes across VSMCs and FRC subsets of EYFP⁺ cells isolated from peripheral 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 peripheral lymph nodes of Ccl19-iEYFP and Ccl19-iEYFP Ltbr^fl/fl mice. (D) Confocal microscopy images showing cross sections of inguinal lymph nodes from Ccl19-iEYFP Ltbr^fl/fl mice. Arrow indicates the localization of Ccl19-tTA⁺ cells in perivascular areas of higher magnification. Microscopy images are representative for three inguinal lymph nodes from three independent experiments. Scale bars: 150 µm (left panels) and 30 µm (right panel). (E and F) Fate analysis of EYFP⁺ cells in inguinal lymph nodes harvested from adult Ccl19-iEYFP⁺ (E) and Ccl19-iEYFP Ltbr^fl/fl (F) mice after Dox administration from E18. High-resolution microscopy images are representative for three inguinal lymph nodes per condition from three independent experiments. Scale bars: 30 µm. (G–I) Quantification of perivascular Ccl19-iEYFP⁺ cells using histology of cross sections from inguinal lymph nodes of fate-mapped Ccl19-iEYFP and Ccl19-iEYFP Ltbr^fl/fl mice (G). Scale bars: 20 µm. (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 inguinal lymph nodes from three independent experiments (H and I).