Fig 2: Internalization of aMSH-PEG-Cy5-C' dots by B16-F10 tumor cells and macrophages. FACS plot of PDPN-PE-Cy7 versus C' dot Cy5 in B16-F10 cells isolated from tumor 4 d after intravenous administration of (A) 50 pmol of aMSH-PEG-Cy5-C' dots or (B) 1% HSA injection. FACS plot of F4/80-PE-Cy7 versus C' dot Cy5 in macrophages isolated from tumor 4 d after intravenous administration of (C) 50 pmol of aMSH-PEG-Cy5-C' dots or (D) 1% HSA injection. FACS plot of F4/80-PE-Cy7 versus C' dot Cy5 in IP tissue macrophages harvested from naive mice 2 d after IP administration of (E) 50 pmol of aMSH-PEG-Cy5-C' dots or (F) 1% HSA injection. FACS analysis of FSC versus C' dot Cy5 at 2 d after introduction of (G) 25 pmol of aMSH-PEG-Cy5-C' dots or (H) 1 × PBS addition to B16-F10 cells in vitro. FACS analysis of FSC versus C' dot Cy5 at 2 d after introduction of (I) 25 pmol of aMSH-PEG-Cy5-C' dots or (J) 1 × PBS addition to wild-type THP-1 cells in vitro. FACS analysis of FSC versus C' dot Cy5 at 2 d after introduction of (K) 25 pmol of aMSH-PEG-Cy5-C' dots or (L) 1 × PBS addition to PMA-differentiated THP-1 cells in vitro. FACS, fluorescence-activated cell sorting; FSC, forward scatter; HSA, human serum albumin; IP, intraperitoneal; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate.

Fig 3: Multiomic transcriptional and epigenetic single-cell analysis of SFs. A Schematic representation of the experimental workflow. We collected ankle synovial tissue from wt and hTNFtg mice, enzymatically disaggregated the tissue, and sorted the cells into one gate representing fibroblasts (CD45-, Ter119-, CD31-, Pdpn+). We profiled the cells with both sc 3' RNA-seq and ATAC-seq using 10X technology and performed scRNA-seq, scATAC-seq, and cross-species integrative analyses with publicly available human RA datasets. B High-quality filtered synovial fibroblasts (n = 5903 for the scRNA-seq and n = 6046 for the sc-ATAC-seq) projected in UMAP space and colored by cluster assignment. C Feature plots on the UMAP embeddings of the SFs shown in B, displaying normalized expression values (for scRNA-seq) and gene activity scores (for scATAC-seq) for Prg4 and Thy1 genes. D Similar to B, but cells are colored by the sample of origin. E scRNA-seq heatmap showing the average scaled expression values for the upregulated genes of each subpopulation (upper panel) and scATAC-seq heatmap of differentially upregulated accessible peaks (lower panel). F Pearson correlation of scaled expression values (RNA) and activity scores (ATAC), followed by hierarchical clustering, for the most variable genes identified in the scRNA-seq analysis

Fig 4: Correlation of DARC expression and known venular markers. a RT-qPCR showing DARC (Darc), P- (Selp), and E-selectins (Sele) expression at the mRNA level in cell-sorted V-EC (CD45-CD31 + gp38-DARC+), NV-EC (CD45-CD31 + gp38-DARC-), and LEC (CD45-CD31 + gp38+) from skin tissue. b RT-qPCR showing Darc, Carbohydrate sulfotransferase 4 (Chst4), and Fucosyltransferase VII (Fut7) expression at the mRNA level in cell-sorted V-EC, NV-EC, and LEC from peripheral lymph nodes (PLN; pool of inguinals, brachials, axillaries, cervicals, and auriculars). c RT-qPCR showing Darc, Chst4, Fut7, and mucosal vascular addressin cell adhesion molecule 1 (Madcam1) expression at the mRNA level in cell-sorted V-EC, NV-EC, and LEC from mesenteric lymph nodes (MLN). Expression data are shown relative to GAPDH of a pool of three experiments for each gene and expressed in arbitrary units (A.U.). Error bars depict mean ± SEM. ns P > 0.05, **P = 0.01, ***P = 0.001. Supporting data values are included in Additional file 2. d–f Flow cytometry contour plots of DARC and peripheral node addressin (PNAd) expression (d), DARC and MAdCAM-1 expression (e), and DARC and ICAM-1 expression (f) in ear skin, PLNs, and MLNs. Cell suspensions were gated on CD45-CD31 + gp38-BEC subset for flow cytometry analysis in all the tissues. n = 3 experiments for each tissue

Fig 5: Lymphoma growth disrupts CCL21 migration cues on FRCs(A) Microscopic analysis of the FRC network (Ccl19RFP) in LN sections. Bottom: examples of FRC sphericity (Ccl19RFP segmentation).(B) Quantification of the FRC density in LN sections of Ccl19RFP mice. n = 7–10 mice per group.(C) Representative images of CCL21 expression in LNs sections of Ccl19RFP mice and gradients of the fluorescence intensity over the distance.(D) CCL21 distribution around and within HEVs (PNAd+) and FRCs (Ccl19RFP).(E) Microscopic analysis of CCL21 (MFI) within masked Ccl19RFP (FRCs) and PNAd+ (HEVs). Control n = 5, tumor n = 7.(F) Immunohistochemistry of CCL21 expression in controls and LNs from diseased tg. Eµ-Myc (n = 7) and Cd19-TAg mice (n = 11); box indicates magnified area. Bar graph depicts percentage of LNs with preserved (pos.) or lost (neg.) CCL21 expression.(G) Proportions of mesenchymal cell subsets (all Ccl19RFP), CD21/CD35+ follicular DCs (FDCs); MadCAM-1+ marginal reticular cells (MRCs); SCA-1+ pericytic reticular cells (PRCs); and T-zone reticular cells (TRCs). n = 4 mice per group.(H and I) FCM quantification of CCL21+ (n = 6–7 mice per group) (H) and PDPN+ expression (n = 7–8 mice per group) (I) in LNs of Ccl19RFP mice.(J and K) Exemplary images (J) and analysis (K) of aSMA (MFI) and CCL21 (integrated density) in a FRC line cultured under static conditions or with laminar flow.Scale bars, 20 µm (A, D, and J) and 100 µm (C and F). Mean and SEM are indicated. Data points represent individual cells (K) or mice (B, E, and G–I). A Mann-Whitney U-test was applied. *p < 0.05; **p < 0.01; ***p < 0.005.