Lymph Node Stromal Cells Underlie the Clinicopathologic Features of Castleman Disease

Castleman disease (CD) is inflammatory lymphoproliferative disorder of unclear etiology. The pathogenesis of the two major subtypes of CD: unicentric CD (UCD) and idiopathic multicentric CD (iMCD), is not known. CD lymph nodes show similar histological features of germinal center depletion, plasmacytosis, increased vascularity and hyalinization, but the molecular basis of these features and their clinical implications are unclear. We aimed to determine the cellular and molecular basis of all subtypes of CD.

The investigation of CD has been constrained by its unpredictable presentation, and lack of cell culture or murine models. To address these limitations, our study leveraged a rare archive of concurrent fresh frozen and FFPE samples obtained from whole lymph node resections. We performed comprehensive multi-omic analysis on nine UCD, iMCD (including TAFRO variant), HHV8-associated MCD cases, and reactive lymph node controls. CODEX proteomic multiplex immunostaining was combined with single nuclei whole transcriptome RNAseq, single nuclei immune repertoire, and bulk DNA mutation analysis. Advanced bioinformatics techniques were employed for an unbiased quantitative analysis of cellular distribution and molecular pathways. Maxfuse was used to integrate proteomic and transcriptomic data. RNAscope ISH was used to confirm the cellular origin of key cytokines.

The immunophenotype and spatial distribution of 645,285 single cells in RLN, 1,768,327 cells in UCD, and 2,071,397 cells in MCD lymph nodes were analyzed. UCD and MCD were characterized by significantly decreased germinal center B cells and increased macrophages, endothelial cells, proliferating plasma cells and fibroblastic reticular stromal cells (FRC) compared to controls. CD21 analysis revealed significantly higher FDC meshworks with greater organization in UCD and MCD. Concentric interdigitation of the activated proliferating FDC cytoplasmic meshworks in between mantle zone B cells was the basis of ‘onion skin’ diagnostic feature of CD and germinal center B cell activation. Microenvironment analysis revealed unique endothelial, plasma cell and mantle zone neighborhoods where non-lymphoid cells closely interacted with lymphoid cells.

We complemented single cell spatial protein expression with single cell gene expression analysis. Single-nuclei transcriptomes for 6,556 cells from RLN, 19,280 from UCD and 24,281 from MCD lymph nodes were analyzed. UCD showed significantly increased non-lymphoid cells and FRC. Differential gene expression and pathway enrichment analyses revealed that FDCs of UCD and MCD exhibited an upregulation of pathways linked to cytoplasmic projections and angiogenesis consistent with activation and proliferation. Other FRC subsets displayed an elevated expression of MAPK, angiogenesis, and extracellular matrix-associated pathways. Expression of key cytokines VEGF and IL-6 was localized to CXCL13+ FDCs, PDGFRA+ T-zone reticular cells (TRC), and ACTA2-positive perivascular reticular cells (PRC) by RNAscope and Maxfuse integration. VEGF expression from FDCs was associated with directional endothelial proliferation towards germinal centers and was the basis of the diagnostic ‘penetrating vasculature/lollipop’ feature of CD. Ligand receptor cell analysis revealed that FDC, TRC and PRC in CD activated JAK-STAT, TGFβ, and MAPK pathways via ligand-receptor interactions involving collagen, integrins, complement components, and VEGF receptors. T, B and plasma cells were polyclonal but showed class-switched and somatically hypermutated IgG1+ plasma cells consistent with stromal cell-driven germinal center activation. HHV8 sequences were noted in plasma cells and cytotoxic T cells from HHV-8-associated MCD case but not other cases. Low levels of PDGFRB c.1997 A>G, p.N666S, ALK c.875G>A, p.R292H and BCOR c.3866G>A, p.G1289D were noted consistent with the proportion of stromal cells.

We performed the largest spatial and single cell characterization of CD lymph node samples and identified the key pathogenic cell types and pathways. We showed that activation and proliferation of specific follicular reticular cells such as FDC, TRC and PRC were associated with VEGF and IL-6 secretion and likely mediate the clinical and pathological features of all subtypes of CD. Collectively, our findings establish that CD as a disorder of aberrant lymph node stromal cells.