Fig. 2: Comparative Analysis of Single Nuclei RNA-Sequencing and Pathway Enrichment in Cell Types of UCD and MCD versus RLN.

A A UMAP plot of 50,117 single nuclei displays identified cell types and their relative abundance per sample, along with global abundance. MCD exhibits significantly increased proliferating plasma cells, cytotoxic and memory T cells, monocyte-macrophages, endothelial-lymphatics, and fibroblastic reticular cells (FRC). UCD demonstrates significantly increased plasmacytoid dendritic cells, plasma cells, and naive B cells. Differential abundance against RLN was tested using a permutation test. Significantly increased proportions are indicated with black triangles, and significantly decreased proportions are indicated with blue triangles. B The differentially expressed genes (DEGs) of UCD and MCD compared to RLN were tested for pathway enrichment using various databases, including Reactome, GO, KEGG, and TRANSFAC. The top significantly enriched pathways for MCD and UCD are displayed. P-value estimates were calculated from a cumulative hypergeometric test⁴⁰. LNSC showed enrichment in pathways related to the extracellular matrix (ECM), cytoplasmic projections, actin cytoskeleton, angiogenesis, MAPK signaling, and VEGF signaling. Macrophages are enriched in inflammatory pathways, whereas plasma cells are enriched in pathways associated with antibody production. C Stromal subsets were annotated by expression of key lineage-defining markers. FDCs are characterized by strong expression of CXCL13, CLU, and FDSP. PRCs show high expression levels of PDGFRB, CCL21, CCL19, ACTA2, TAGLN, and CXCL12. TRCs show high CCL21 and CXCL12 expression. BECs show high CDH5, ENG, and PECAM1, while LECs show high PROX1. D. UMAPs of merged stromal subsets from cases and controls. TRCs, PRCs, BECs, and LECs are predominantly from MCD. BRCs and FDCs are predominantly from UCD. E SCT normalized IL-6 module score (IL-6, OSMR, IL-6ST, LIFR) expression is displayed in the top row while VEGFA categorized by stromal cell and sample type is shown in the bottom row. CRCs, PRCs, and FDCs of CD show high IL-6 module and VEGF expression. Gene expression significance versus RLN was calculated using Wilcoxon signed-rank test, and module score significance versus RLN was calculated using Tukey's Honest Significance Difference. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Source data are provided as a Source Data file in the supplementary dataset.