Single-cell transcriptomic sequencing identifies nodal endothelial and FRCs expressing IL-6–related genes. (A) UMAP plot of 25 445 cells from the affected lymph node of Twin-CS demonstrating distinct clusters by cell types, with a majority of B cells, followed by T/NK cells, plasma cells, and others. (B) Violin plot showing the average expression of IL-6 pathway genes (IL6, IL6ST, OSMR, and LIFR) was strongest in the nodal endothelial cells and fibroblasts (2-tailed P < .000001; Kruskal-Wallis test). (C) UMAP plot of 406 cells identified as fibroblasts and endothelial cells from the affected lymph node of Twin-CS showing 4 distinct clusters, with the fibroblast compartment further segregated into FRCs, follicular dendritic cells (FDCs), and myofibroblasts. (D) Violin plot showing the average expression of IL-6 pathway genes were most prominent in FRCs and endothelial cells (2-tailed P < .000001; Kruskal-Wallis test). (E) Cell-type annotation on the spatial enhanced resolution omics sequencing (Stereo-seq) bin50 data encompassing the same cell types identified on single-cell RNA-sequencing (scRNAseq) analysis of affected lymph node of Twin-CS. (F) Enlarged region showing enrichment of endothelial cells corresponding to vascular structures (circled in cyan), whereas follicle structures (circled in yellow) were enriched in B cells and FDCs, and surrounded by FRCs, endothelial cells, and other cell types. (G) After a cell segmentation approach (bin 20, 15 μm diameter), cell-type specific markers similarly annotated them to their expected regions on the spatial map. (H) The proportion of cells expressing IL-6–pathway genes (IL6, IL6ST, OSMR, and LIFR) above the threshold (Q3, third quartile) within each cell type as annotated on bin50 data as shown in panel E. (I) Lymph node tissue of Twin-CS demonstrated IL-6 and IL-6ST coexpression with endothelial cells (CD31⁺) and FRCs (CCL21⁺) on double immunofluorescence (scale bars, 20 μm). NK cell, natural killer cell; UMAP, uniform manifold approximation and projection.