Terranova-Barberio, M. | bioRxiv Science

bioRxiv2025

Addressing complex autofluorescence signatures in solid tissue samples to enhance full spectrum flow cytometry of non-immune cells.

FSFC is an emerging technology that can greatly enhance our understanding of the single-cell proteomic landscape. However, its application to cells derived from solid tissues has been hampered by their complex autofluorescence signatures and lack of optimized tools for non-immune cells. Here, we present a protocol and discuss key controls that minimize the impact of unmixing errors enabling us to resolve multiple EC subpopulations isolated from different tissues in models of chronic tissue injury. Research Topic(s)Vascular biology, cell heterogeneity, full spectrum flow cytometry Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/695385v2_ufig1.gif" ALT="Figure 1000"> View larger version (43K): org.highwire.dtl.DTLVardef@1745181org.highwire.dtl.DTLVardef@1930db9org.highwire.dtl.DTLVardef@16a0b3dorg.highwire.dtl.DTLVardef@107ec29_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsOptimisation of a FSFC panel to enable in-depth phenotyping of tissue- and model-specific endothelial subpopulations from solid tissues. Discussion of appropriate controls to minimize the impact of tissue autofluorescence and enhance the signal-to-noise ratio for cell phenotyping in complex models of inflammation and fibrosis. Trajectory analysis to track cellular plasticity over time. Application of full spectrum cell sorting to isolate rare endothelial subpopulations with complex phenotypes.

cell biology↗

bioRxiv2024

Jagged-1+ Tregs Mediate Lymphatic Remodeling in Tumour-Draining Lymph Nodes

The tumour-draining lymph nodes (tdLNs) are crucial sites for early immune surveillance of tumours. During tumour development, tdLNs undergo significant stromal remodelling that impacts lymph node (LN) architecture. The reconstruction of tdLNs is dependent on the crosstalk between regulatory T cells (Tregs) and resident stromal cells. The Notch ligand Jagged1 (Jag1), highly expressed in skin Tregs, is critical for facilitating stem cell-mediated tissue regeneration. However, the role of Jag1pos Tregs in mediating the activity of tdLN-resident immune cells and stromal cells remains unexplored. Here, we used the B16-F10 tumour model to assess the role of Jag1pos Tregs during tdLN development. During tumor progression, late-stage tdLN Tregs expressed higher levels of Jag1 than early-stage tdLNs. Conditional deletion of Jag1 in Tregs markedly restrained tdLN expansion, without influencing effector T cell abundance or activation profile. Transcriptomic analysis of tdLNs revealed downregulation of lymphatic endothelial cell (LEC)-related markers in mice with Treg-specific Jag1 ablation. Disruption of lymphatic networks in the tdLN was further confirmed by flow cytometric profiling and histological analysis. In vivo permeability assessment by FITC-dextran demonstrated perturbed lymphatic drainage, likely due to suppressed LN lymphangiogenesis. Finally, we identify the presence of Jag1pos Tregs in tdLNs of human melanoma patients. Overall, our results highlight that crosstalk between Jag1pos Tregs and LECs is required for lymphatic sinus expansion, which is critical for the formation of the tdLN niche during melanoma progression.

immunology↗