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Lymphoid organ stromal cells comprise different subsets whose origin remains unknown. Herein, we exploit a genetic lineage-tracing approach to show that splenic fibroblastic reticular cells (FRCs), follicular dendritic cells (FDCs), marginal reticular cells (MRCs), and mural cells, but not endothelial cells, originated from embryonic mesenchymal progenitors of the Nkx2-5+Islet1+ lineage. This lineage included embryonic mesenchymal cells with lymphoid tissue organizer (LTo) activity capable of supporting ectopic lymphoid-like structures, and a subset of resident spleen stromal cells that proliferated and regenerated the splenic stromal microenvironment following resolution of a viral infection. These findings identify progenitor cells that generate stromal diversity in spleen development and repair, and suggest the existence of multipotent stromal progenitors in the adult spleen with regenerative capacity.

There is an exponentially growing interest in targeting immune checkpoint molecules in breast cancer (BC), particularly in the triple-negative subtype where unmet treatment needs remain. This study was designed to analyze the expression, localization, and prognostic role of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, and TIM3 in primary BC. Gene expression analysis using the METABRIC microarray dataset found that all six immune checkpoint molecules are highly expressed in basal-like and HER2-enriched compared to the other BC molecular subtypes. Flow cytometric analysis of fresh tissue homogenates from untreated primary tumors show that PD-1 is principally expressed on CD4+ or CD8+ T cells and CTLA-4 is expressed on CD4+ T cells. The global proportion of PD-L1+, PD-L2+, LAG3+, and TIM3+ tumor-infiltrating lymphocytes (TIL) was low and detectable in only a small number of tumors. Immunohistochemically staining fixed tissues from the same tumors was employed to score TIL and tertiary lymphoid structures (TLS). PD-L1+, PD-L2+, LAG3+, and TIM3+ cells were detected in some TLS in a pattern that resembles secondary lymphoid organs. This observation suggests that TLS are important sites of immune activation and regulation, particularly in tumors with extensive baseline immune infiltration. Significantly improved overall survival was correlated with PD-1 expression in the HER2-enriched and PD-L1 or CTLA-4 expression in basal-like BC. PD-1 and CTLA-4 proteins were most frequently detected on TIL, which supports the correlations observed between their gene expression and improved long-term outcome in basal-like and HER2-enriched BC. PD-L1 expression by tumor or immune cells is uncommon in BC. Overall, the data presented here distinguish PD-1 as a marker of T cell activity in both the T and B cell areas of BC associated TLS. We found that immune checkpoint molecule expression parallels the extent of TIL and TLS, although there is a noteworthy amount of heterogeneity between tumors even within the same molecular subtype. These data indicate that assessing the levels of immune checkpoint molecule expression in an individual patient has important implications for the success of therapeutically targeting them in BC.

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