Niche-associated heterogeneity of lymphoid stromal cells

Stromal cells establish the compartmentalization of lymphoid tissues critical for the immune response. However, the full diversity of lymph node stromal cells remains undefined. Germinal centers (GCs) form in the center of follicles during a T-dependent immune response and are comprised of two niches, the light zone, supported by the stromal follicular dendritic cells (FDCs), and the dark zone (DZ), recently described to contain stromal Cxcl12-expressing reticular cells (CRCs). GC B cells must cycle to the DZ to achieve efficient antibody affinity maturation, but the properties of CRCs in the DZ are relatively unexplored. Here we find CRCs are present in GC DZs consistently across lymphoid tissues with network morphology and surface marker phenotype distinct from FDCs. CRCs also form smaller networks in the T-zone proximal side of primary follicles. Real-time two-photon microscopy revealed GC B cells explore CRC networks suggesting CRCs may support DZ GC B cell activities through cell-cell interactions. To further explore the heterogeneity of non-endothelial stromal cells supporting LN niches, we used droplet-based single-cell RNA sequencing and identified transcriptional profiles for 8 niche-associated stromal subsets. We found subsets of T-zone reticular cells (TRCs), marginal reticular cells, FDCs and perivascular cells, which have been shown to support the T-zone, subcapsular sinus, follicles and blood vessels, respectively. We also identified and localized new stromal subsets; Ch25h+ Ccl19lo TRCs at the T-zone perimeter, Cxcl9+ TRCs in the T-zone, CD34+ stromal cells in the capsule and medullary vessel adventitia and Inmt+ stromal cells enriched in the medullary cords. Additionally, we validated Tmem119 and Pthlh as novel FDC markers and Sox9 as a novel marker of FDCs and CRCs. These transcriptional profiles enable exploration of niche-associated stromal functions, stromal development and the context-dependent activities of lymphocytes in an immune response