Single-cell omics and flow cytometry identify distinct immune states of dural and brain-infiltrating IL-17-producing γδ T cells after experimental stroke.

backgroundγδ T cells boost inflammatory responses and exacerbate tissue damage after ischemic stroke. However, the origin, dynamics, and tissue adaptation of γδ T cells in the ischemic brain and its border regions remain poorly understood. A systematic integration of large-scale datasets is urgently needed. Here, we investigated the impact of ischemic stroke on the state of meningeal and brain-infiltrating γδ T cells and explored their potential contributions to post-stroke inflammation.

methodsWe conducted an integrated analysis of publicly available single-cell RNA sequencing (scRNA-seq) datasets, which included meningeal and brain-infiltrating Ptprc+ (CD45+) immune cells following experimental stroke. γδ T cells were identified and subsequently classified into distinct subtypes through data integration and reference mapping. Subtype-specific functions, tissue residency signatures, migratory programs, and the cellular interactions between γδ T cells and endothelial cells or fibroblasts in the dura and brain were investigated, respectively. Key findings were validated by flow cytometry and immunofluorescence assays in vivo.

resultsOn day 2 post-experimental stroke, the number of parenchymal γδ T cells significantly increased while dural γδ T cells decreased. The majority of γδ T cells residing in the meninges and infiltrating the brain, both under homeostatic conditions and following stroke, were Rorc⁺ and belonged to the Vγ6⁺ γδ17 cell subset. Compared to dural γδ T cells, brain-infiltrating γδ T cells showed reduced tissue residency capacities, higher migratory pathway activation, and lower Ki‑67 positivity, indicating acute recruitment. In contrast, dural γδ T cells exhibited greater IL‑17-producing capacities on day 3. Redistributions of dural γδ T cells were analyzed, and immunofluorescence revealed a close spatial association between dural γδ T cells and CD31+ cells. Cell–cell communication analysis predicted increased interactions between γδ T cells and CD45− cells in both the dura and the brain.

conclusionOur data indicate that most meningeal and brain-infiltrating γδ T cells after stroke share an activated γδ17 phenotype but display compartmentalized dynamics in activation, proliferation, and migration. These results establish a foundation for further studies on the spatially distinct roles of γδ T cells in post-stroke immunity.