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

Overview

1. Department of Neurology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany
2. Department of Neurology, First Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang, China
3. Department of Neurology, the First Affiliated Hospital of Soochow University, Jiangsu, China

Abstract

Background: γδ 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.

Methods: We 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.

Results: On 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.

Conclusion: Our 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.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12974-026-03794-3.

Reproduced under the paper's license (CC BY), from the paper cited above.

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Data

Datasets cited

• figshare:32048876, at figshare; found in DataCite
• figshare:32048882, at figshare; found in DataCite
• figshare:32048885, at figshare; found in DataCite
• geo:GSE191075, at NCBI GEO; found in “Introduction to the publicly available…”
• github.com/xmc811/scillus, at github.com; found in the references

Data availability

The codes generated for this study are shown in the Additional file 1. All information on the mice used in this study is shown in Additional file 2. The processed flow cytometric results are attached to the Additional file 3. All publicly available single-cell RNA sequencing datasets analyzed in this study were retrieved from the Gene Expression Omnibus database as provided by the original studies, which were also mentioned in the Methods section. The integrated scRNAseq data and in-house γδ T cells used in the current study are available from the Google Drive (Link: https://drive.google.com/drive/folders/1G5p2NjEPxWSxETw0hckLma45-9xYKylg?usp=sharing). The dataset supporting the conclusions of this article is included within the article and its additional files.

Reproduced under the paper's license (CC BY), from the paper cited above.

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Recorded: type, language, journal, volume, issue, pages, dates, 8 authors, 6 keywords, 12 MeSH terms, 3 funders, 54 references, 27 RRIDs.

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This paper

Zha, M., Jander, A., Cai, H., Piepke, M., Degenhardt, K., Winter, L., Magnus, T., & Gelderblom, M. (2026). Single-cell omics and flow cytometry identify distinct immune states of dural and brain-infiltrating IL-17-producing γδ T cells after experimental stroke. Journal of neuroinflammation, 23(1), 127. https://doi.org/10.1186/s12974-026-03794-3

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