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This research investigates the divergent roles of DNT subsets in ischaemic stroke, particularly focusing on their neurotoxic and protective mechanisms.

May 8, 2026

Abstract Number: Esoc2026a1962 Dichotomous Roles of DNT Subsets in Ischaemic Stroke: Tcrγδ+ Drives Neurotoxicity While Tcrαβ+ Mediates Perforin-Dependent Immunoregulation

Key Points

This research investigates the divergent roles of DNT subsets in ischaemic stroke, particularly focusing on their neurotoxic and protective mechanisms.
Performed single-cell RNA sequencing and flow cytometry on murine dMCAO models and patient PBMCs.
Utilized CXCR3-/- and perforin knockout mouse models to examine DNT functions.
Conducted adoptive transfer of TCRαβ+ DNTs into Rag1-/- or wild-type mice to evaluate therapeutic potential.
Identified TCRγδ+ DNTs as neurotoxic through CXCR6/IL-17A pathway, with their elimination improving prognosis.
TCRαβ+ DNTs promoted immunoregulation via CXCR3 and perforin, showing protective effects against neurotoxicity.
Higher TCRγδ/TCRαβ ratios in patients correlated with worse NIHSS scores.

Abstract

Abstract Background and aims CD3+CD4−CD8− double-negative T cells (DNTs) are an unconventional T cell population traditionally implicated in immune tolerance and reported to exert regulatory T cell-like functions. While DNTs accumulate in the ischaemic brain, whether they are neuroprotectors or effectors is debated.Our preliminary RNA sequencing reveals brain-accumulating DNTs comprise distinct TCRαβ+ and TCRγδ+ subsets with divergent survival and chemokine profiles. Hypothesising this heterogeneity underlies current controversies, we aimed to elucidate their specific mechanisms. Methods We utilised single-cell RNA sequencing and flow cytometry on murine dMCAO models and patient PBMCs. Mechanisms were dissected using Cxcr3-/-, Prf1-/- (perforin knockout), and Prf1-GFP mice; therapeutic potential was evaluated via adoptive transfer into Rag1-/- or wild-type mice. Results We identified two axes. 1) Pathogenic: TCRγδ+ DNTs—the dominant, apoptosis-resistant subset—infiltrated via CXCR6 and secreted IL-17A, exacerbating neurotoxicity; their elimination improved prognosis. 2)Protective: TCRαβ+ DNTs were recruited via the CXCL9-11/CXCR3 axis (abrogated in Cxcr3−/−). Unlike the neurotoxic subset, TCRαβ+ cells exerted immunoregulatory effects via Perforin. Prf1-GFP tracing revealed DNT-derived perforin specifically eliminated pro-inflammatory leukocytes. Crucially, adoptive transfer of WT TCRαβ+ DNTs reduced infarct volume and improved recovery, whereas Prf1-/- DNTs failed to provide protection. Clinically, higher TCRγδ/TCRαβ ratios correlated with worse NIHSS scores. Conclusions This study defines a dichotomous DNT landscape: pathogenic TCRγδ+ cells (CXCR6/IL-17A) versus protective TCRαβ+ cells (CXCR3/Perforin). We propose TCRαβ+ DNTs as a novel adoptive cell therapy that utilizes "regulatory cytotoxicity" to resolve post-stroke inflammation. Conflict of interest Xiaoyi Jiang, Zhiyu Leng, Ming Yuan, Qinxue Sun, Yuhualei Pan, Gang Li: nothing to disclose Figure 1 - belongs to Conclusions

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Cite This Study

Jiang et al. (Fri,) studied this question.

synapsesocial.com/papers/69fd7e90bfa21ec5bbf06c14 https://doi.org/https://doi.org/10.1093/esj/aakag023.918

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