Abstract: Lymph node (LN) function requires the organization of cells into higher order spatial units. However, the principles governing LN architecture in health and disease remain poorly understood. Here, we utilized single-cell and spatial mapping to interrogate the mechanisms directing immune cell organization in human LNs and its disruption in architecturally distinct lymphoma entities: indolent follicular lymphoma (FL) and aggressive diffuse large B cell lymphoma (DLBCL). Our data substantiate the central role of LN-resident stromal cells in chemokine-driven lymphocyte zonation and reveal an inflammatory feedback loop fueled by tumor-reactive T cells, triggering stromal remodeling, progressive loss of homeostatic chemokine gradients and tissue organization from non-malignant to FL and DLBCL. Loss of homeostatic chemokines was associated with adverse patient survival, identifying the underlying architectural rearrangement as a key event during lymphomagenesis. Collectively, our results highlight the principles of LN organization and suggest how lymphoma-induced microenvironmental reprogramming drives loss of tissue organization. Data overview: Seurat object for mIF datamIF_seurat_final.rds Seurat object for scRNA-seq lymph node datascRNA_LN_stromal_cells.rds Seurat object for spatial transcriptomics/Xenium dataxenium_merged_seurat.rds Raw molecule-level transcript coordinates Xenium outputs per sampleXenium_transcripts_FL.parquetXenium_transcripts_DLBCL.parquet Files related to Figure 3 - TCR-seq analysis202402_cd8_TCRobj_mapped.rds202402_TCRrepertoire.rds Files related to Figure 3 - SCENIC/GRN analysisGRN_90pt.rds Files related to Figure 3 - Seurat object for scRNA-seq co-culture dataCoCulture_DLBCL.rds Files related to Figure 4 - Scriabin analysisinteraction_potential.rdsmIF_knn_k6_pct_totalinteractions.csvmIF_knn_k6_pct_totalinteractions_rLN.csv Files related to Figure 5 - Bulk transcriptomicscibersortx_signature_matrix.txt