The normal human lymph node cell classification and landscape defined by high-dimensional spatial proteomic

To comprehensively classify in situ all cells in the human lymph node (LN) and provide spatial and mutual relational data for its constituents, we employed a 78-marker hyperplexed staining method, MILAN, and an analytical bioinformatic pipeline, BRAQUE, to characterize 19 LN samples free of pathology. We classified 77 cell types encompassing T, B, innate immune and stromal cells. By leveraging the expression profiles of TCF7 and co-inhibitory receptors distribution, CD4 and CD8 T-cells were classified into 27 subsets with distinct phenotypes and tissue locations. CD5 and TCF7 expression defined novel B-cell types. CD27+ mature B-cells occupied a novel nodal spaces non-overlapping with the cortex and the plasma-cell rich medullary cords. Type 2 conventional dendritic cells were located in nodular paracortical aggregates. Statistically controlled pairwise neighborhood analysis showed sparse cell-cell interactions, known and new neighbors, established and novel LN landscape niches, which should prompt new insight into LN physiology.