Identification and mapping of distinct human lymph node stromal cell subsets by combining single cell RNA sequencing with spatial transcriptomics Cristoforo Grasso1,2, Catarina Gago de Graça3, Johanna Semmelink1,2, Ester Remmerswaal5, Perry Moerland4, Aldo Jongejan4, Reina E. Mebius3, Lisa G. M. van Baarsen1,2. 1. Department of Rheumatology and Clinical Immunology and Department of Experimental Immunology, Amsterdam Infection and Immunity Institute, Amsterdam UMC and University of Amsterdam, Amsterdam, the Netherland 2. Amsterdam Rheumatology and Immunology Center (ARC), Academic Medical Center, Amsterdam, the Netherlands 3. Department of Molecular Cell Biology and Immunology, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam Infection and Immunity Institute, Amsterdam, the Netherlands 4. Amsterdam UMC, University of Amsterdam, Bioinformatics Laboratory, Department of Epidemiology and Data Science, Biostatistics and Bioinformatics 5. Department of Experimental Immunology, Amsterdam Infection and Immunity Institute, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands. Lymph node stromal cells (LNSCs) constitute a crucial immunomodulatory role in human health and disease. Here, we report the profiling of 12,000 LNSCs isolated from a human lymph node (LN) and analysed by droplet-based single-cell RNA sequencing. Uniform manifold approximation and projection analysis (UMAP) revealed 10 major subtypes of human non-endothelial stromal cells (SCs), 4 subtypes of lymphatic endothelial cells (LECs) and 4 subtypes of blood endothelial cells (BECs). Interestingly, detailed SC analysis shed light on unprecedented heterogeneity in human LN fibroblasts. This study comprehensively defined the gene signatures of the 10 identified SC subtypes: NR4A1+ SC, CCL21+ medullary reticular cells, CCL19+ T- zone reticular cells, CD34+ CXCL14+SC, pericytes, desmin+ medullary reticular cells, LAMP5+ and HLA-DR+ SCs being active in antigen processing and presentation, SEPT7+ smooth muscle cells and GLDN+ neurofibroblasts. To localize each distinct subtype within a human LN we transferred the SC-subset-specific gene signatures to a publicly available human spatial transcriptomic data set of a human LN. The results underpin the distinct location of the different identified LNSC subtypes. This study determined the human LNSC landscape and sets the stage for future investigations to learn more about the relationship between their LN niche and immunomodulatory function during health and disease.