Th1 cells are critical tissue organizers of myeloid-rich perivascular activation niches. Th1 cells are critical tissue organizers of myeloid-rich perivascular activation niches

Aggregating immune cells within perivascular niches (PVN) can regulate tissue immunity in infection, autoimmunity and cancer. How cells are assembled at PVNs and the activation signals imparted within remain unclear. Here, we integrate dynamic time-resolved in vivo imaging with a novel spatially-resolved platform for microanatomical interrogation of transcriptome, immune phenotype and inflammatory mediators in skin PVNs. We uncover a complex positive-feedback loop within CXCL10+PVNs that regulates myeloid and Th1 cell positioning for exchange of critical signals for Th1 activation. Th1 cells spend ∼24h in the PVN, receiving initial peripheral activation signals, before redeploying to the inflamed dermal parenchyma. Niche-enriched, CCR2-dependent myeloid cells were critical for Th1 IFNγ-production. In turn, PVN instructional signals enabled Th1s to orchestrate PVN assembly by CXCR2-dependent intra-tissue myeloid cell aggregation. The results reveal a critical tissue organizing role for Th1s, gained rapidly on tissue entry, that could be exploited to boost regional immunity. Overall design: We integrated IV-MPM single cell dynamics with spatially resolved single cell transcriptomics, flow cytometry and immuno-protein profiling to identify key regulators in the choreography and functional programming of lymphocytes specifically in the perivascular niche (PVN). REX3 (Reporter for the EXpression of Cxcr3 ligands) mice were immunized with CFA/OVA and OT-II Th1 cells adoptively transferred, on d5 post-immunization we imaged the tissue by IV-MPM for CXCL10+ aggregates located perivascularly, based on acute CD31 staining immediately prior to imaging. The spatial coordinates for CXCL10+ PVN (niche) and non-niche regions of then inflamed ear pinna were mapped using the microscope user interface. We optimized the size of the biopsy to ensure full niche capture from the imaging coordinates, with a 2mm punch biopsy providing sufficient tissue margins around the PVN for accurate niche isolation, and often capturing 2-3 adjacent niches in the same biopsy. Non-niche regions were identified in a similar fashion, ensuring the absence of CXCL10+ PVN aggregates. Niche-enriched and non-niche region biopsies were analyzed for spatially-restricted differences using single-cell RNA sequencing